Polypeptides having anti-senescent effects and uses thereof

ABSTRACT

Polypeptides which can provide a senotherapeutic effect are provided herein. The polypeptides can be formulated for topical application and can be applied topically to a subject to provide a senotherapeutic effect in the subject or in the cells of the subject.

CROSS REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/877,164, filed Jul. 22, 2019, which is entirelyincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 27, 2020, isnamed 56292-701_201.txt and is 10,604 bytes in size.

BACKGROUND

Accumulation of cellular senescence is not only a product of organismaging, it can also actively contribute to further senescence inductionin a positive feedback cycle. Among the hallmarks of aging, cellularsenescence may occupy a central position, integrating primary,antagonistic and integrative aspects of aging. First, senescence cancompromise tissue-repair and renewal capacity of the affected tissue,since it decreases proliferation capacity of progenitor cells. Second,senescent cells can alter the paracrine signaling milieu, beingcharacterized by their senescence-associated secretory phenotype (SASP),which can induce inflammation and further cellular senescence, possiblyexacerbating a potentially deleterious inflammatory response andpossibly promoting tissue injury. Third, the accumulation of senescentcells with age has been documented in several tissues, including, butnot limited to, the skin. Fourth, cellular senescence can be an activeplayer in diseases, such as macular degeneration, dementia,atherosclerosis and cancer.

Senescent cells can be identified by Senescence-Associatedbeta-galactosidase (SA-BGal) production, p16 expression, and alphathalassemia/mental retardation X-linked chromatin remodeling protein(ATRX) foci accumulation in the nuclei. Functional alterations alsodistinguish senescent cells, including decreased proliferation capacityand resistance to mitogenic stimuli.

SUMMARY

Described herein are compositions comprising an isolated, synthetic,and/or recombinant polypeptide comprising an amino acid sequence of LKGI(SEQ ID NO: 5) or analogs thereof, an amino acid sequence of LKGIL (SEQID NO: 6) or analogs thereof, or an amino acid sequence of WLKGI (SEQ IDNO: 7) or analogs thereof. In some embodiments, such polypeptide cancomprise at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 amino acids.Alternatively or additionally, such polypeptide comprises at most 100,90, 80, 70, 60, 50, 40, 35, 30, 25, or 20 amino acids. In someinstances, the polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NO:1-4.

Also described herein are isolated, synthetic and/or recombinantpolypeptides comprising an amino acid sequence of SEQ ID NO:8, which isrepresented by X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀ or an analog thereof

-   1. wherein X₁ is E, X₂ is T, X₄ is K, X₆ is W, X₇ is L, X₉ is G, and    X₁₀ is I; and (i) X₃ is not S (SEQ ID NO: 8); or-   2. (ii) if X₅ is any amino acid then X₈ is not G (SEQ ID NO: 29); or-   3. (iii) if X₈ is any amino acid then X₅ is not N (SEQ ID NO: 30);-   4. or (iv) any one of (i), (ii), or (iii) optionally with 1, 2, 3,    or 4 conservative amino acid substitutions; or-   5. (b) the amino acid sequence has at least 70%, 80%, 85%, 90%, or    95% identity to a second sequence SEQ ID NO:2 wherein X₁ is A, X₂ is    T, X₃ is A, X₄ is K, X₅ is A, X₆ is W, X₇ is L, X₈ is K, X₉ is G,    and X₁₀ is I, optionally with 1, 2, 3, or 4 conservative amino acid    substitutions; or (c) the amino acid sequence has at least 70%, 80%,    85%, 90%, or 95% identity to a third sequence SEQ ID NO:3 wherein X₁    is K, X₂ is L, X₅ is I, X₆ is L, X₈ is G, and X₁₀ is A; and (i) if    X₉ is any amino acid then X₃ is not N (SEQ ID NO: 31); or (ii) if X₃    is any amino acid then X₉ is not S (SEQ ID NO: 32); or (iii) if X₄    is any amino acid then X₇ is not L (SEQ ID NO: 33); or (iv) if X₇ is    any amino acid then X₄ is not S (SEQ ID NO: 34), and; or (v) any one    of (i), (ii), (iii), or (iv) optionally with 1, 2, 3, or 4    conservative amino acid substitutions; or (d) the amino acid    sequence has at least 70%, 80%, 85%, 90%, or 95% identity to a    fourth sequence SEQ ID NO:4 wherein X₁ is W, X₂ is L, X₃ is K, X₄ is    G, X₅ is I, X₆ is L, X₇ is R, X₈ is E, X₉ is A, and X₁₀ iS A,    optionally with 1, 2, 3, or 4 conservative amino acid substitutions.

In some embodiments, the amino acid sequence comprises LKGI (SEQ IDNO:5). In some embodiments, the amino acid sequence comprises WLKGI (SEQID NO:7). In some embodiments, the amino acid sequence comprises LKGIL(SEQ ID NO:6). In some embodiments, the amino acid sequence has at least70%, 80%, 85%, 90%, or 95% identity to a sequence of SEQ ID NO:1. Insome embodiments, the amino acid sequence is SEQ ID NO:1. In someembodiments, the amino acid sequence is SEQ ID NO:2. In someembodiments, the amino acid sequence has at least 70%, 80%, 85%, 90%, or95% identity to a sequence of SEQ ID NO:3. In some embodiments, theamino acid sequence is SEQ ID NO:3. In some embodiments, the amino acidsequence is SEQ ID NO:4. In some embodiments, the recombinantpolypeptide comprises at least 10 amino acids, 15 amino acids, or 20amino acids.

The compositions provided herein can be formulated for use as atherapeutic, nutraceutical, or cosmetic.

In some embodiments, the formulation further comprises a therapeutic,nutraceutical, or cosmetic excipient. In some embodiments, the excipientis configured for topical application. In some embodiments, theexcipient is configured as a topical supplement. In further embodiments,the formulation is configured for application to human skin. In someembodiments, the formulation is a cream, a transdermal patch, a topicalpatch, an ointment, an oil, a gel, a liquid, a powder, a lotion, aserum, an emulsion, a moisturizer, a toner, a foam, a face mask, amousse, an aerosol, a spray, a cleanser, a hydrogel patch, a powder, ora shampoo. In some embodiments, the formulation may be used inconjunction with a sonic treatment, an ultrasonic treatment, a LEDtreatment, a light treatment, an electrical treatment, or aradiofrequency treatment. In some embodiments, the transdermal patchdelivers the formulation to the epidermal layer of the skin. In someembodiments, the transdermal patch delivers the formulation to theepidermal and dermal layers of the skin. In some embodiments, theformulation is delivered in minimum or low amounts systemically in thesubject or is not intended to be delivered directly into the bloodstreamof the subject. In some embodiments, the formulation acts locally at andnear the delivery site. In some embodiments, the formulation has minimalto no effects systemically.

In some embodiments, the formulation is configured as an ediblesupplement. In some cases, the formulation is configured as a beverage.

Described herein are therapeutic, nutraceutical, or cosmeticformulations comprising at least one recombinant or syntheticpolypeptide herein and a therapeutic, nutraceutical, or cosmeticexcipient.

In some embodiments, the excipient is configured for topicalapplication. In some embodiments, the excipient is configured as atopical supplement. In further embodiments, the formulation isformulated for application to human skin. More specifically, theformulation can be configured to penetrate topically from the epidermisto the dermis. In some embodiments, the formulation can be configured topenetrate topically through the epidermis and dermis layers. In someembodiments, the formulation can be configured to penetrate topicallythrough the epidermis layer and have low penetration into the dermislayer. Often, the penetration of a component in a formulation may beassessed using various permeation studies, including but not limited tothose using a Franz diffusion cell. In some embodiments, the formulationcomprises a carrier, a microsphere, a liposome, or a micelle in order tocarry the polypeptide and control the release time and/or penetrationdepth of the polypeptide through the skin. In some cases, a formulationherein is a cream, an ointment, a gel, a liquid, an oil, a powder, alotion, a serum, an emulsion, a moisturizer, a foam, a face mask, amousse, an aerosol, a spray, a cleanser, a toner, a topical patch, ahydrogel patch, or a shampoo.

In some embodiments, the formulation is configured as an ediblesupplement. In some embodiments, the formulation is configured as abeverage. In some embodiments, the formulation is configured as atablet, a capsule, a gel, a gummy, or a powder.

Described herein are methods of treating a condition in a subject inneed thereof, the method comprising administering to the subject atherapeutic, nutraceutical, or cosmetic formulation comprising an aminoacid sequence of at least one of SEQ ID NOS:5-7.

In some embodiments, the administering comprises topically applying theformulation to the subject. In further embodiments, the subject is ahuman or other animal. In some embodiments, the method comprisesadministering an effective amount of the formulation to the subject.

In some embodiments, the condition is a disorder associated withaccumulation of senescent cells in the subject. In some embodiments, thedisorder associated with accumulation of senescent cells comprises agingskin. In some embodiments, the condition is a disorder associated withprogeria and/or an effect of progeria. In some embodiments, progeriacomprises conditions having premature aging symptoms in the epidermaland dermal layers of skin.

Described herein are methods of reducing cellular senescence in asubject in need thereof, the method comprising administering to thesubject a therapeutic, nutraceutical, or cosmetic formulation comprisinga polypeptide comprising an amino acid sequence of LKGI (SEQ ID NO:5)optionally with 1 conservative amino acid substitution.

In some embodiments, the polypeptide comprises at least 4 amino acids,10 amino acids, 15 amino acids, or 20 amino acids. In some embodiments,the formulation comprises an amino acid sequence of WLKGI (SEQ ID NO:7)optionally with 1 conservative amino acid substitution. In someembodiments, the formulation comprises an amino acid sequence of LKGIL(SEQ ID NO:6) optionally with 1 conservative amino acid substitution. Insome embodiments, the polypeptide comprises at least 5 amino acids, 10amino acids, 15 amino acids, or 20 amino acids. In some embodiments, thepolypeptide comprises no more than 10 amino acids, 15 amino acids, 20amino acids, 25 amino acids, 30 amino acids, 35 amino acids, or 40 aminoacids.

In some embodiments, the formulation further comprises a therapeutic,nutraceutical, or cosmetic excipient.

Described herein are methods of reducing cellular senescence in asubject in need thereof, the method comprising administering to thesubject a therapeutic, nutraceutical, or cosmetic formulation comprisingat least one polypeptide described herein.

In some embodiments, the formulation further comprises a therapeutic,nutraceutical, or cosmetic excipient. In some embodiments, theadministering comprises applying the formulation to a portion of theskin of the subject. In some embodiments, the formulation extends alifespan of a plurality of cells of the subject, induces SIRT6expression in a plurality of cells of the subject, increases cellrenewal rates in a plurality of cells of the subject, promotes apoptosisin a plurality of cells of the subject, promotes DNA repair in aplurality of cells of the subject, increases collagen production in aplurality of cells of the subject, increases hyaluronic synthaseproduction in a plurality of cells of the subject, decreases ATRXnuclear foci accumulation in a plurality of cells of the subject,decreases p16 expression in a plurality of cells of the subject,decreases senescence associated beta-galactosidase production in aplurality of cells of the subject, decreases IL8 expression in aplurality of cells of the subject, decreases MMP1 expression in aplurality of cells of the subject, increases BLM expression in aplurality of cells of the subject, and/or prevents UV-induced DNA damagein a plurality of cells of the subject.

Described herein are methods of treating a condition in a subject inneed thereof, the method comprising administering to the subject atherapeutic, nutraceutical, or cosmetic formulation comprising an aminoacid sequence of SEQ ID NO:5 optionally with 1 conservative amino acidsubstitution.

In some embodiments, the therapeutic, nutraceutical, or cosmeticformulation comprises an amino acid sequence of SEQ ID NO:6 optionallywith 1 conservative amino acid substitution. In some embodiments, thetherapeutic, nutraceutical, or cosmetic formulation comprises an aminoacid sequence of SEQ ID NO:7 optionally with 1 conservative amino acidsubstitution.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates the effects of individual polypeptides on progeriafibroblast cell number and senescence level.

FIG. 2 illustrates the senotherapeutic effects of four polypeptides,Peptide 14 (panel A), Peptide 13 (panel B), Peptide 15 (panel C), andPeptide 16 (panel D), on senescent fibroblasts. *p<0.05; **p<0.01;***p<0.001.

FIG. 3 illustrates a senotherapeutic effect of a polypeptide whichpromotes a higher number of cells with less ATRX foci/nuclei (panel A),a lower average of ATRX foci/nuclei (panel B), and a higher number ofcells with less than 10 ATRX foci/nuclei (panel C). *p<0.05; **p<0.01.

FIG. 4 illustrates the effect of a senotherapeutic polypeptide which candecrease the number of senescent fibroblasts in a cell population duringa 3-week long exposure keeping the senotherapeutic effect for at leastone week after treatment (panel A) without inducing cell toxicity orsignificantly affecting cellular proliferation during this period (panelB). ***p<0.001; ****p<0.0001.

FIG. 5 illustrates that treatment with a polypeptide can promote a doseresponse decrease in cellular senescence, as measured by average ATRXfoci accumulation per cell in cells derived from multiple donors.

FIG. 6 illustrates that cellular senescence can be induced infibroblasts with exposure to etoposide (panel A), treatment ofetoposide-induced senescent cells with a polypeptide can result inreduced senescence (panel B), cellular senescence can be induced by UVBexposure (panel C), and treatment of a UVB treated sample with apolypeptide can result in reduced senescence (panel D). *p<0.05;**p<0.01; ***p<0.001.

FIG. 7 illustrates that human skin equivalents treated with apolypeptide can show higher quality according to an overall structureanalysis score (panel A), that human skin equivalents treated with apolypeptide can comprise significantly fewer senescent cells thanuntreated human skin equivalents (panels A and B), and altered geneexpression in which p16 can have a significantly lower expression inpolypeptide treated epidermis and dermis, compared to untreated control;and IL-8 and MMP-1 were significantly less expressed inpolypeptide-treated dermis, compared to untreated counterparts (panelC). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

FIG. 8 illustrates pAkt S473 can be significantly decreased in bothepidermal and dermal samples treated with a polypeptide (panel A),polypeptide treatment can decrease SA-B gal staining in UVB exposedsamples indicating protection from UVB-induced cellular senescence(panel B), and that a polypeptide can increase the expression of SIRT6and BLM (panel C). *p<0.05; **p<0.01; ***p<0.001.

FIG. 9 illustrates increased epidermal layer thickness in human skinequivalents as histological images (panel A) and statistical analysis ofacquired data (panel B). **p<0.01.

FIG. 10 illustrates the predicted three dimensional structures of twopolypeptides, Peptide 14 (panel A) and Peptide 13 (panel B), as well asthe superposition of the two polypeptides (panel C).

FIG. 11A illustrates hematoxylin and eosin (H&E) stained histologicalimages of the 3D skin equivalents (top row) and ex vivo skin biopsysamples (bottom row) cultured with no Peptide 14 (Control) and 12.5 μMPeptide 14 (12.5 μM Pep 14) for 5 days.

FIG. 11B illustrates the predicted age, also referred to as molecularDNA age, of the 3D skin model (top graph) and ex vivo skin biopsy(bottom graph) samples treated with 12.5 μM Peptide 14 (treatment) waslower than the predicted age for samples that were untreated control(ctrl). **p<0.01.

FIG. 11C illustrates the predicted age of ex vivo skin biopsy treatedwith 12.5 μM Peptide 14 (12.5 μM Pep 14) was lower than the predictedage of samples that were untreated control (ctrl).

FIG. 12A illustrates the relative melanin content of cellular pellet ofMeWo cells treated with the positive control of isobutyl methylxanthine(IBMX), negative control, Peptide 14 with IBMX, and retinoic acid withIBMX for 7 days after 14 day pre-treatment culture with IBMX. Data arepresented as average and standard deviation of melanin contentnormalized to negative control (100%). **p<0.01; ***p<0.001;****p<0.0001.

FIG. 12B illustrates the relative melanin content of cell culture mediumsupernatant from MeWo cells treated with the positive control with IBMX,negative control, Peptide 14, and retinoic acid for 7 days after 14 daypre-treatment culture with isobutyl methylxanthine (IBMX). Data arepresented as average and standard deviation of melanin contentnormalized to negative control (100%). *p<0.05; **p<0.01.

FIG. 13A illustrates the relative melanin content of cellular pellet ofMeWo cells treated with the positive control of IBMX, negative control,Peptide 14 with IBXM for 14 days, retinoic acid with IBMX for 14 days,Peptide 14 with IBXM for 7 days, retinoic acid with IBMX for 7 days,after 14 day pre-treatment culture with isobutyl methylxanthine (IBMX).Data are presented as average and standard deviation of melanin contentnormalized to negative control (100%). *p<0.05; **p<0.01; ****p<0.0001.

FIG. 13B illustrates the relative melanin content of cell culture mediumsupernatant from MeWo cells treated with the positive control of IBMX,negative control, Peptide 14 with IBXM for 14 days, retinoic acid withIBMX for 14 days, Peptide 14 with IBXM for 7 days, retinoic acid withIBMX for 7 days, after 14 day pre-treatment culture with isobutylmethylxanthine (IBMX). Data are presented as average and standarddeviation of melanin content normalized to negative control (100%).*p<0.05; **p<0.01; ****p<0.0001.

FIG. 14A illustrates the relative mRNA expression level of tyrosinase ofMeWo cells treated with the positive control of IBMX, negative control,Peptide 14 with IBXM for 14 days, retinoic acid with IBMX for 14 days,Peptide 14 with IBXM for 7 days, retinoic acid with IBMX for 7 days.Data are presented as average and standard deviation of 2-ddCtnormalized to GAPDH and negative control expression (100%). *p<0.05;**p<0.01; ****p<0.0001.

FIG. 14B illustrates the relative mRNA expression level of melanocyteinducing transcription factor (MITF) of MeWo cells treated with thepositive control of IBMX, negative control, Peptide 14 with IBXM for 14days, retinoic acid with IBMX for 14 days, Peptide 14 with IBXM for 7days, retinoic acid with IBMX for 7 days. Data are presented as averageand standard deviation of 2-ddCt normalized to GAPDH and negativecontrol expression (100%). *p<0.05; **p<0.01; ***p<0.001.

FIG. 14B illustrates the relative mRNA expression level of dopachrometautomerase (DCT) of MeWo cells treated with the positive control ofIBMX, negative control, Peptide 14 with IBXM for 14 days, retinoic acidwith IBMX for 14 days, Peptide 14 with IBXM for 7 days, retinoic acidwith IBMX for 7 days. Data are presented as average and standarddeviation of 2-ddCt normalized to GAPDH and negative control expression(100%). **p<0.01; ***p<0.001; ****p<0.0001.

FIG. 15A illustrates the H&E-stained histological images of the in vitrohuman skin models treated with a vehicle only (control), Peptide 13, orPeptide 14.

FIG. 15B illustrates the mean of histology scores of human skin modelstreated with a vehicle only (control), Peptide 14, or Peptide 13, whichwere 21.00, 23.83, and 23.44, respectively. **p<0.01.

FIG. 16 illustrates an example of the left side of the face treated withPeptide 14 at baseline (left, Baseline) before treatment and after 12weeks of treatment (right, 12 weeks).

FIG. 17 illustrates relative mRNA expression levels of p16, BLIMP1,ZYG11B, IL-8, Ki-67, ZIC1, MMP1, HAS2 of the epidermal and dermal layersof 3D skin equivalents treated with a control, Peptide 14, Peptide 13,or Retinoic Acid. Data are presented as 2-ddCt normalized to GAPDH anduntreated control. *p<0.05.

FIG. 18 illustrates prolonged life and healthspan of Caenorhabditiselegans (C. elegans) as acquired data indicating that treatment witheither 1 μM or 2 μM polypeptide improved worm thrashing (panel A),pumping (panel B), and median lifespan (panel C). *p<0.05; **p<0.01.

FIG. 19 illustrates the effect of polypeptide sequence LKGIL (SEQ IDNO:6) (A, B, C), and polypeptide sequence WLKGI (SEQ ID NO:7) (D, E, F)to decrease cellular senescence without promoting cell death. In panelsA and D, the y-axis indicates the relative senescence level normalizedto untreated control. In panels B and D, y-axis indicates the relativecell number normalized to untreated control. In panels C and F, y-axisindicates the average ATRX foci accumulation per cell. *p<0.05;**p<0.01.

DETAILED DESCRIPTION

Aging can largely result from a functional decline in the ability tomaintain tissue homeostasis and integrity, possibly coupled withdiminished responses to physiological demands under conditions ofstress.

In a mosaic model of skin aging, senescent cells can be induced byintrinsic and extrinsic stimuli, such as time/age, UV exposure andsmoking, among others. According to this model, senescent cellsaccumulate in the skin and actively promote tissue aging by altering thelocal microenvironment through a senescence-associated secretoryphenotype (SASP) composed of proinflammatory cytokines among others. Insome cases, senescent cells further promote skin aging by compromisingepidermal stem cell renewal and promoting senescence of otherwise normalcells. Therefore, senescent cells are not only a product of skin aging,but are also active players in the aging process.

Skin dysfunction can affect the development of organismal aging andage-associated diseases and disorders. For instance, skin health andregular barrier function can be associated with lower levels ofinflammatory and age-related cytokines IL-10 and IL-6 compared tocounterparts with a compromised skin barrier. Increased levels of IL-1(3and IL-6 have been observed in the serum of patients with severalage-associated disorders, including cardiovascular disease (CVD),Alzheimer's disease, and type II diabetes. In some cases, in olderadults' serum IL-6 can be associated with all-cause mortality, CVD,cancer, and liver-related mortality. In some cases, the recovery ofepidermal function can effectively reduce circulating TNFα, IL-10 andIL-6 cytokine levels.

Provided herein are polypeptides, compositions comprising polypeptidesand other components, and methods of use thereof. The polypeptides andcompositions comprising the polypeptides can provide an anti-senescenteffect (e.g., on cells of a subject). Polypeptides can promote adecrease of senescence levels in cells and tissues by promotingapoptosis, promoting DNA repair, and/or inhibiting DNA-damage inducedsenescence. Anti-senescent effects can include, but are not limited to,increased cell renewal rates, increased collagen production, increasedhyaluronan synthase production, decreased ATRX nuclear fociaccumulation, decreased p16 expression, lower SASP production, decreasedsenescence-associated beta-galactosidase production, reduced unevenpigmentation, maintenance of or improvement in an epidermal barrier, andreduced transepidermal water loss (TEWL). The polypeptides andcompositions comprising the polypeptides provided herein may inhibit,prevent, or slow aging-associated and/or senescence-associated diseasesor conditions. Furthermore, the polypeptides and compositions comprisingthe polypeptides may enhance or improve healthspan and/or promotelifespan.

It is to be understood that the methods and compositions describedherein are not limited to the particular methodology, protocols, andreagents described herein and as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the methods and compositions described herein, which will belimited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the inventions described herein belong. Although anymethods, devices, and materials similar or equivalent to those describedherein can be used in the practice or testing of the invention describedherein, the preferred methods, devices, and materials are now described.

In a peptide or polypeptide, suitable conservative substitutions ofamino acids are known to those of skill in this art and can be madegenerally without altering the biological activity of the resultingmolecule. Watson et al. (1987, Molecular Biology of the Gene, 4thEdition, The Benjamin Cummings Pub. Co., p. 224) is incorporated hereinby reference. The amino acids may be either in the L- or D-isomericform. When an amino acid residue is part of a polypeptide chain, theD-isomeric form of the amino acid can be substituted for the L-aminoacid residue, as long as the desired functional property is retained.The amino acids herein can be represented by their standard IUPAC1-letter code or 3-letter code. An amino acid residue represented by “X”or “Xxx” refers to any one of the naturally occurring or non-naturallyoccurring amino acid residues known in the art or to a modification of anearby residue. Amino acid substitutions are typically of singleresidues, such substitutions are preferably made with those set forth inTable 1, but may be of multiple residues, either clustered or dispersed.An amino acid can be replaced with a different naturally occurring or anon-conventional amino acid residue. Such substitutions may beclassified as “conservative,” in which case an amino acid residuecontained in a polypeptide is replaced with another naturally occurringamino acid of similar character either in relation to polarity, sidechain functionality, or size. Additions encompass the addition of one ormore naturally occurring or non-conventional amino acid residues.Deletion encompasses the deletion of one or more amino acid residues.

TABLE 1 Conservative and Non-Conservative Amino Acid SubstitutionsConservative Non-Conservative Amino Acid Substitutions Substitutions AD, E, G, S, and T P and V C G, R, S, W, and Y F D A, E, G, H, N, V, andY E A, D, G, K, Q, and V F I, L, and Y C, S, and V G A, C, D, E, and RS, V, and W H D, L, N, P, Q, R, and Y I F, L, M, N, and V K, R, S, and TK E, M, N, Q, R, and T I L F, H, I, M, P, Q, R, V, S and W M I, K, L, R,T, and V N D, H, I, K, S, T, and Y P H, L, Q, R, and S A and T Q E, H,K, L, P, and R R C, G, H, K, L, M, P, Q, I and S T, and W S A, C, N, P,T, W, and Y F, G, I, L, and R T A, K, M, N, R, and S I and P V D, E, I,L, and M A, F, and G W C, L, R, and S G Y C, D, F, H, N, and S

Substitutions encompassed by the present disclosure may also be“non-conservative,” in which an amino acid residue which is present in apolypeptide is substituted with an amino acid having differentproperties, such as a naturally-occurring amino acid from a differentgroup (e.g., substituting a charged or hydrophobic amino acid withalanine), or alternatively, in which a naturally-occurring amino acid issubstituted with a non-conventional amino acid.

The term “analog(s)” as used herein refers to a composition that retainsthe same structure or function (e.g., binding to a receptor) as apolypeptide, such as the same protein from a different organism.Examples of analogs include mimetics or peptidomimetics, peptides, smalland large organic or inorganic compounds, as well as derivatives andvariants of a polypeptide herein. Such derivatives and variants refer topolypeptides that differ from the naturally occurring polypeptides byone or more amino acid deletions, additions, substitutions, orside-chain modifications. In some embodiments, a peptide analog is apeptide in which one or more of the amino acids has undergone side-chainmodifications. Examples of side-chain modifications contemplated by thepresent disclosure include modifications of amino groups such as byreductive alkylation by reaction with an aldehyde followed by reductionwith NaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonicacid (TNBS); acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5-phosphate followed by reduction with NaBH₄. In someembodiments, a peptide analog is one in which the guanidine group ofarginine residue(s) is modified by the formation of heterocycliccondensation products with reagents such as 2,3-butanedione,phenylglyoxal and glyoxal; carboxyl group(s) is modified by carbodiimideactivation via O-acylisourea formation followed by subsequentderivatization, for example, to a corresponding amide; sulphydrylgroup(s) may be modified by methods such as carboxymethylation withiodoacetic acid or iodoacetamide; performic acid oxidation to cysteicacid; formation of mixed disulphides with other thiol compounds;reaction with maleimide, maleic anhydride or other substitutedmaleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH. In any of theanalogs herein, any modification of cysteine residues preferably do notaffect the ability of the peptide to form the necessary disulphidebonds. In some embodiments, a peptide analog comprises tryptophanresidue(s) that are modified, for example, by oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides; tyrosine residuesaltered by nitration with tetranitromethane to form a 3-nitrotyrosinederivative; imidazole ring(s) of a histidine residue modificationaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate; proline residue(s)modified by, for example, hydroxylation in the 4-position; glycosylationvariants from a completely unglycosylated molecule to a modifiedglycosylated molecule; and altered glycosylation patterns as a resultfrom expression of recombinant molecules in different host cells.

The term “isolated” means altered from its natural state; i.e., if itoccurs in nature, it has been changed or removed from its originalenvironment, or both. For example, a naturally occurring polynucleotideor a polypeptide naturally present in a living animal in its naturalstate is not “isolated,” but the same polynucleotide or polypeptideseparated from the coexisting materials of its natural state is“isolated,” as the term is employed herein.

The terms “protein,” “peptide,” “oligopeptide,” or “polypeptide” as usedherein refer to any composition that includes two or more amino acidsjoined together by a peptide bond. It will be appreciated thatpolypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids, and thatmany amino acids, including the terminal amino acids, may be modified ina given polypeptide, either by natural processes such as glycosylationand other post-translational modifications, or by chemical modificationtechniques which are well known in the art. The known modifications,which may be present in polypeptides of the present disclosure, include,but are not limited to, acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of a flavonoid or a heme moiety, covalentattachment of a polynucleotide or polynucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycation, glycosylation, glycosylphosphatidylinositol (GPI) membrane anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to polypeptides such asarginylation and ubiquitination. The term “protein” also includes“artificial proteins” which refers to linear or non-linear polypeptides,consisting of alternating repeats of a polypeptide (e.g., SEQ IDNOs:1-7) and a spacer. A DNA construct encoding the polypeptide andspacer alternate repeats can be synthesized using methods known in theart (see Rotzschke et al., 1997, Proc. Natl. Acad. Sci. USA94:14642-14647).

The term “purified” as used herein to describe a polypeptide,polynucleotide, or other composition, refers to such polypeptide,polynucleotide, or other composition separated from one or morecompounds which are usually associated with it in nature. Such othercompositions can be, for example, other polypeptides or polynucleotides,carbohydrates, lipids, etc. The term “purified” can also be used tospecify the separation of monomeric polypeptides of the disclosure fromoligomeric forms such as homo- or hetero-dimers, trimers, etc. Asubstantially pure polypeptide typically comprises at least about 50%,60%, 70%, 80%, or 90% weight/weight of a polypeptide sample, or morepreferably at least about 95%, 96%, 97%, 98%, 99%, or 99.5%weight/weight of a polypeptide sample. As a preferred embodiment, apolypeptide of the present disclosure is at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% pure relativeto heterologous polypeptides.

The term “subject,” or “patient” as used herein, encompasses animals. Insome embodiments, the subject may be a mammal. Examples of mammalsinclude, but are not limited to, any member of the Mammalian class:humans, non-human primates such as chimpanzees, and other apes andmonkey species; farm animals such as cattle, horses, sheep, goats, andswine; domestic animals such as rabbits, dogs, and cats; laboratoryanimals including rodents, such as rats, mice, and guinea pigs, and thelike. The mammal can be a human.

The terms “treat,” “treating,” or “treatment,” as used herein, includedelaying the onset of, reducing the occurrence of, or ameliorating atleast one symptom of a disease or condition, preventing additionalsymptoms, inhibiting the disease or condition, e.g., arresting thedevelopment of the disease or condition, relieving the disease orcondition, causing regression of the disease or condition, relieving acondition caused by the disease or condition, or stopping the symptomsof the disease or condition either prophylactically and/ortherapeutically.

The term “therapeutically acceptable,” as used herein, refers to amaterial, including but not limited, to a salt, carrier, or diluent,which does not abrogate the biological activity or properties of thecompound, and is relatively nontoxic, i.e., the material may beadministered to a subject without causing undesirable biological effectsor interacting in a deleterious manner with any of the components of thecomposition in which it is contained.

The term “carrier,” as used herein, refers to relatively nontoxicchemical compounds or agents that facilitate the incorporation of acompound into cells or tissues.

The term “diluent,” as used herein, refers to chemical compounds thatare used to dilute the compound of interest prior to delivery. Diluentscan also be used to stabilize compounds because they can provide a morestable environment. Salts dissolved in buffered solutions (which alsocan provide pH control or maintenance) are utilized as diluents in theart, including, but not limited to, a phosphate buffered salinesolution.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent or a compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disease or condition being treated. The result can bereduction and/or alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Forexample, an “effective amount” for therapeutic uses is the amount of thecomposition comprising a compound as disclosed herein required toprovide a clinically significant decrease in disease symptoms. Anappropriate “effective” amount in any individual case may be determinedusing techniques, such as a dose escalation study.

Senescent cells can be identified by senescence-associatedbeta-galactosidase (SA-BGal) production, p16 expression, presentation ofSASP, and/or alpha thalassemia/mental retardation X-linked chromatinremodeling protein (ATRX) foci accumulation in the nuclei. Functionalalterations that can also distinguish senescent cells, include, but arenot limited to, decreased proliferation capacity and resistance tomitogenic stimuli.

Aging generally results from a functional decline in the ability tomaintain tissue homeostasis and integrity, coupled with diminishedresponses to physiological demands under conditions of stress.

With regard to skin aging, a mosaic model has been proposed, in whichsenescent cells are induced by intrinsic and extrinsic stimuli, such astime/age, UV exposure, and smoking, among other stimuli. According tothe mosaic model, senescent cells accumulate in the skin and activelypromote tissue aging by altering the local microenvironment through asenescence-associated secretory phenotype (SASP) composed ofproinflammatory cytokines among others. It has been shown that senescentcells can promote skin aging by compromising epidermal stem cell renewaland promoting senescence of otherwise normal cells. Therefore, senescentcells may not only be a product of skin aging, but may also be an activeplayer in the aging process.

Polypeptides have properties such as multifunctional behavior which canmake them useful for cosmetic or therapeutic applications, includingsenotherapy. Skin dysfunction observed during aging can affect thedevelopment of age-associated diseases and disorders.

Polypeptides

Polypeptides and compositions comprising polypeptides as provided hereincan provide a senotherapeutic effect, e.g., the polypeptide can reducesenescence, such as by halting senescence, preventing senescence,inhibiting senescence, reversing senescence, destroying senescent cells,killing senescent cells, removing senescent cells, or by any suitablemechanism of reducing the burden or effects of senescent cellaccumulation. Such polypeptides can in some cases comprise the aminoacid sequence LKGI (SEQ ID NO:5). Compositions comprising suchpolypeptides can be employed or used to provide a senotherapeuticeffect.

The polypeptides (e.g., senotherapeutic polypeptides) can comprise atleast 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40 or more amino acids. In some cases, the polypeptides may be no morethan 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids long.In some cases, the polypeptides can be from 4 to 25, from 4 to 15, orfrom 4 to 10 amino acids in length. In some embodiments, thepolypeptides can comprise at least 30, 40, 50, 60, 70, 80, 90, 100, ormore amino acids. In certain embodiments, the polypeptides may compriseless than 100, 90, 80, 70, 60, 50, 40, 30, or fewer amino acids.

Examples of polypeptides which can provide a senotherapeutic effect areprovided in Table 2 below.

TABLE 2 Example Polypeptide Amino Acid Sequences Name SEQ ID NO:Amino Acid Sequence Peptide 14 1 ETAKHWLKGI Peptide 13 2 ATAKAWLKGIPeptide 15 3 KLKGILRGAA Peptide 16 4 WLKGILREAA

A polypeptide can be an isolated or recombinant polypeptide, which cancomprise an amino acid sequence of X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀. Amino acidsincluded in the polypeptide can comprise a natural amino acid, which caninclude alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N),aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Glu, Q), glutamicacid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile,I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M),phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine(Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), or valine (Val, V).

In some cases, the isolated or recombinant polypeptide comprising anamino acid sequence of X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀ can comprise an amino acidsequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity to afirst sequence, ETAKHWLKGI (SEQ ID NO:1), wherein X₁ is E, X₂ is T, X₄is K, X₆ is W, X₇ is L, X₉ is G, and X₁₀ is I; and wherein at leasteither (i) X₃ is not S (SEQ ID NO: 8); or (ii) if X₅ is any amino acidthen X₈ is not G (SEQ ID NO: 29); or (iii) if X₈ is any amino acid thenX₅ is not N (SEQ ID NO: 30); or (iv) any one of (i), (ii), or (iii),where the sequence can optionally comprise 1, 2, 3, or 4 conservativeamino acid substitutions. In some cases, the isolated or recombinantpolypeptide can include an amino acid sequence having at least 70%, 75%,80%, 85%, 90%, 95%, or 100% identity to a sequence of ETAKHWLKGI (SEQ IDNO:1). The present disclosure also contemplates analogs, such aspeptidomimetics of the above.

In some cases, the isolated or recombinant polypeptide comprising anamino acid sequence of X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀ can comprise an amino acidsequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity to asecond sequence, ATAKAWLKGI (SEQ ID NO:2), wherein X₁ is A, X₂ is T, X₃is A, X₄ is K, X₅ is A, X₆ is W, X₇ is L, X₈ is K, X₉ is G, and X₁₀ isI. Such a recombinant polypeptide can optionally comprise 1, 2, 3, or 4conservative amino acid substitutions. In some cases, the isolated orrecombinant polypeptide can include an amino acid sequence having atleast 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to a sequence ofATAKAWLKGI (SEQ ID NO:2). The present disclosure also contemplatesanalogs, such as peptidomimetics of the above.

In some cases, the isolated or recombinant polypeptide comprising anamino acid sequence of X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀ can comprise an amino acidsequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity to athird sequence, KLKGILRGAA (SEQ ID NO:3), wherein at least either (i) ifX₉ is any amino acid then X₃ is not N (SEQ ID NO: 31); or (ii) if X₃ isany amino acid then X₉ is not S (SEQ ID NO: 32); or (iii) if X₄ is anyamino acid then X₇ is not L (SEQ ID NO: 33); or (iv) if X₇ is any aminoacid then X₄ is not S (SEQ ID NO: 34); or (v) any one of (i), (ii),(iii), or (iv), where the sequence can optionally comprise 1, 2, 3, or 4conservative amino acid substitutions. In some cases, the isolated orrecombinant polypeptide can include an amino acid sequence having atleast 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to a sequence ofKLKGILRGAA (SEQ ID NO:3). The present disclosure also contemplatesanalogs, such as peptidomimetics of the above.

In some cases, the isolated or recombinant polypeptide comprising anamino acid sequence of X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀ can comprise an amino acidsequence having at least 80%, 85%, 90%, or 95% identity to a fourthsequence, WLKGILREAA (SEQ ID NO:4), wherein X₁ is W, X₂ is L, X₃ is K,X₄ is G, X₅ is I, X₆ is L, X₇ is R, X₈ is E, X₉ is A, and X₁₀ is A. Sucha recombinant polypeptide can optionally comprise 1, 2, 3, or 4conservative amino acid substitutions. In some cases, the isolated orrecombinant polypeptide can include an amino acid sequence having atleast 80%, 85%, 90%, 95%, or 100% identity to a sequence of WLKGILREAA(SEQ ID NO:4). The present disclosure also contemplates analogs, such aspeptidomimetics of the above.

In some cases, a polypeptide can comprise the amino acid sequence LKGI(SEQ ID NO:5), LKGIL (SEQ ID NO:6), or WLKGI (SEQ ID NO:7) (see Table 3below).

TABLE 3 Example Polypeptide Amino Acid Sequences SEQ ID NO:Amino Acid Sequence 5 LKGI 6 LKGIL 7 WLKGI

A polypeptide comprising one of SEQ ID NOs:5-7 may comprise at least 4,5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 amino acids. In some embodiments, apolypeptide comprising one of SEQ ID NOs:5-7 may comprise no more than4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 aminoacids. SEQ ID NOs:5-7 can be at the N-terminus of a polypeptide, at theC-terminus of a polypeptide, or between the N-terminus and C-terminus ofa polypeptide. In some cases, a polypeptide can comprise more than oneof SEQ ID NOs:5-7.

The polypeptide can be isolated, substantially pure, or purified. Insome cases, an isolated polypeptide can be (i) synthesized chemically or(ii) expressed in a host cell and purified away from associated andcontaminating proteins. In some cases, the polypeptide can be present ina host cell as the expression product of a portion of an expressionvector, and can be linked to a protein moiety or linked to a chemicalmoiety.

Analogs, including peptidomimetics, of the disclosed polypeptides canprovide a senotherapeutic effect. The peptides and polypeptidesdisclosed herein may include peptidomimetic equivalents.

In some cases, as discussed above, a polypeptide can have a sequenceidentity to a polypeptide described herein. Sequence identity of apolypeptide can refer to an exact amino acid-to-amino acidcorrespondence of two polypeptide sequences. In some cases, techniquesfor determining sequence identity can include determining the amino acidsequence and comparing the amino acid sequence to a second amino acidsequence. Two or more sequences can be compared by determining theirpercent identity, or the number of exact matches between two alignedsequences divided by the length of the longer sequence and multiplied by100. Percent identity may also be determined, for example, by comparingsequence information using the advanced BLAST computer program,including, e.g., version 2.2.9, available from the National Institutesof Health. The BLAST program is based on the alignment method of Karlinand Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990) and asdiscussed in Altschul, et al., J. Mol. Biol., 215:403-410 (1990); Karlinand Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993); andAltschul et al., Nucleic Acids Res. 25:3389-3402 (1997). The program maybe used to determine percent identity over the entire length of thepolypeptides being compared. Default parameters are provided to optimizesearches with short query sequences in, for example, the blastp program.The program also allows the use of an SEG filter to mask-off segments ofthe query sequences as determined by the SEG program of Wootton andFederhen, Computers and Chemistry 17:149-163 (1993).

Compositions

Disclosed herein are compositions comprising one or more of thepolypeptides described herein. In some embodiments, the compositions maybe senotherapeutic. In some cases, a composition can be used to treat anage-related disease or condition or age-associated disorder, e.g., todelay the onset of, reduce the occurrence of, or ameliorate theage-related disease or condition or age-associated disorder. In somecases, a composition can be used to treat tissue lesion, to delay theonset of, reduce the occurrence of, or ameliorate the tissue lesion, forexample, UV damage upon sun exposure.

In some cases, a composition may include, e.g., an effective amount of apolypeptide, alone or in combination, with one or more vehicles (e.g.,therapeutically acceptable compositions or therapeutically acceptablecarriers) and other therapeutically effective compounds. In someembodiments, the effective amount of the polypeptide refers to having adesired effect on a subject, including but not limited to a cell, atissue, or an organism, treated with the composition. In someembodiments, the effective amount of the polypeptide has minimal or loweffect systemically on the treated subject. In some embodiments, theeffective amount of the polypeptide has maximal effect locally at ornear the treated area. In some embodiments, the formulation can beconfigured to penetrate topically from the epidermis to the dermis. Insome embodiments, the formulation is configured to penetrate topicallythrough the epidermis layers. In some embodiments, the effective amountof a polypeptide is at least 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1μM, at least 10 μM, at least 25 μM, at least 50 μM, at least 75 μM, atleast 100 μM, at least 150 μM, at least 200 μM, at least 250 μM, atleast 300 μM, at least 350 μM, at least 400 μM, at least 450 μM or atleast 500 μM. In some instances, the effective amount of a polypeptideis between about 1 nM to about 1000 nM, about 5 nM to about 750 nM,about 25 nM to about 750 nM, or about 50 nM to about 500 nM. In someinstances, the effective amount of a polypeptide is between about 1 μMto about 500 μM, about 25 μM to about 250 μM, about 50 μM to about 200μM, or about 75 μM to about 125 μM. In some instances, the effectiveamount of a polypeptide is at least 0.00001%, 0.00005%, 0.0001%,0.0005%, 0.001%, at least 0.005%, at least 0.01%, at least 0.05%, atleast 0.1%, at least 0.5%, at least 1%, at least 1.5%, at least 2%, atleast 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, orat least 5% (w/w) of the final composition. In some instances, theeffective amount of a polypeptide between about 0.00001% to about 5%,0.00001% to about 1%, 0.00001% to about 0.1%, about 0.001% to about 5%,about 0.005% to about 4%, about 0.005% to about 3%, about 0.005% toabout 2%, about 0.005% to about 1%, or about 0.005% to about 0.5% of thefinal composition In some embodiments, the effective amount of thepolypeptide for an in vivo application may be at least 2, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500times than the amount used in an in vitro application. The effectiveamount of polypeptide results in some dermis penetration of thepolypeptide, in some instances about 1% penetration, about 2%penetration, about 4% penetration, about 5% penetration, or about 10%penetration. In some instances, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, or 10% of the polypeptide in the composition applied onto theskin penetrates into the dermis. In some instances, at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, or 10% of the polypeptide in the compositionapplied onto the skin penetrates into the dermis. In some instances, theamount used in an in vivo application is a factor of amount of dermalpenetration in an in vitro penetration study. In some instances, thefactor for the amount used in an in vivo application is at least 2, 5,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 times theamount of in vitro dermal penetration.

In some cases, the compositions described herein can be administeredwith one or more additional nutraceutical, cosmetic, therapeutic, orpharmaceutical treatments (e.g., co-administered, sequentiallyadministered, or co-formulated).

In some embodiments, the formulation may be used in conjunction with oneor more treatments. In some embodiments, the formulation may be usedwith a sonic treatment, an ultrasonic treatment, a LED treatment, alight treatment, an electrical treatment, a radiofrequency treatment, orother dermatological treatments. In some embodiments, the composition isapplied to the skin before, after, or during the treatment.

A composition can be formulated for topical application. For example,the composition may be formulated for application onto skin. In someembodiments, the composition is configured as a topical supplement.Formulations such as those for topical application can be a cream, anointment, a gel, a liquid, a powder, a lotion, a serum, an emulsion, amoisturizer, a foam, a face mask, a mousse, an aerosol, a spray, acleanser, a toner, a topical patch, a hydrogel patch, or a shampoo.Polypeptides applied topically can be applied to an affected area, to anarea which may become affected in the future, a portion of the subject,or substantially the entire subject. In some cases, a topical treatmentcan be applied with a buffer, another topical treatment, a cream, or amoisturizer.

A composition, such as for topical application, can be formulated as acosmetic composition. Examples of cosmetic compositions can includemakeup, foundation, sunscreen, after sun lotion, and skin care products,including anti-aging skin care products. In some cases, makeupcompositions can leave color on the face, and can include foundation,bronzer, mascara, concealer, eye liner, brow color, eye shadow, blusher,lip color, powder, a solid emulsion compact, or other makeup items. Insome cases, skin care products can be those used to treat or care for,or somehow moisturize, improve, accelerate renewal, protect, preventdamage, or clean the skin. A skin-care product can be applied as acream, a topical patch, a hydrogel patch, a transdermal patch, anointment, a gel, a liquid, a powder, a lotion, a serum, an emulsion, anoil, a clay, a moisturizer, a foam, a face mask, a mousse, an aerosol, aspray, a cleanser, a toner, or a shampoo. In some cases, skin-careproducts can be in the form of an adhesive, a bandage, exfoliant, atoothpaste, a moisturizer, a lotion, a primer, a lipstick, a lip balm,an anhydrous occlusive moisturizer, an antiperspirant, a deodorant, apersonal cleansing product, an occlusive drug delivery patch, a nailpolish, a powder, a tissue, a wipe, a hair conditioner, or a shavingcream.

A composition as contemplated herein can also be edible, i.e.,formulated as an edible supplement or beverage, such that thecomposition is formulated to be safely consumed by humans. In somecases, an edible composition can be therapeutically effective to treatan age-related disease or condition or age-associated disorder. In somecases, an edible supplement can be configured as a tablet, capsule,chew, gummy, powder, food bar, meal replacement bar, or a food additive.In some cases, a beverage can be formulated to comprise water, a soda, atea, coffee, milk, a juice, a shake, a drink, or other edible liquid.

In some cases, a composition can comprise a skin conditioning agent(e.g., a humectant, exfoliant, emollient, or hydrator). A humectant canbe for moisturizing, reducing scaling, or stimulating removal ofbuilt-up scale from the skin. An exfoliant can be for the removal of oldskin cells from the surface, and can be a physical exfoliant or achemical exfoliant. An emollient can be a preparation or ingredientwhich can soften dry, rough, or flakey skin. A hydrator can be formoisturizing, reducing scaling, or stimulating removal of built-up scalefrom the skin. In some cases, emollient is an agent that prevents waterloss and has a softening and soothing effect on skin. In someembodiments, emollients may comprise at least one of plant oils, mineraloil, shea butter, cocoa butter, petrolatum, fatty acids (animal oils,including emu, mink, and lanolin), triglycerides, benzoates, myristates,palmitates, stearates, glycolipids, phospholipids, squalene, glycerin,rose hip oil, andiroba oil, grape seed oil, avocado oil, plum seed oil,pracaxi oil, Calycophyllum spruceanum oil, almond oil, argan oil,caprylic/capric triglyceride, jojoba butter, jojoba oil, Spectrastat G2,ceramide, and algae extract. In some cases, the composition comprises askin hydrating agent, also referred to as a skin hydrator. In somecases, the skin hydrating agent include but are not limited to glycerin,squalene, sorbitol, hyaluronic acid, hyaluronic acid derivatives, sodiumhyaluronate, sodium hyaluronate crosspolymer, niacinamide,glycoproteins, pyrrolidone carboxylic acid (PCA), lysine HCl, allantoinand algae extract. In some embodiments, the composition comprises atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% skin conditioningagent. In some embodiments, the composition comprises about 1% to about70%, about 1% to about 60%, about 1% to about 50%, about 5% to about50%, about 5% to 45%, or about 5% to 40% skin conditioning agent.

A composition can comprise a shine control agent, which can improve orregulate the shiny appearance of skin. Shine control agents can beporous in nature. Such agents can provide a reservoir to absorb excessmoisture to reduce the appearance of shine. Shine control agents can besilicas, magnesium aluminum silicates, talc, sericite and variousorganic copolymers. Particularly effective shine control agents caninclude silicates or carbonates that are formed by reaction of acarbonate or silicate with the alkali (IA) metals, alkaline earth (IA)metals, or transition metals, and silicas (silicon dioxide). Preferredshine control agents are selected from the group consisting of calciumsilicates, amorphous silicas, calcium carbonates, magnesium carbonates,zinc carbonates, bentonite clay, and combinations thereof.

A composition can comprise a film forming agent, which can aid filmsubstantivity and adhesion to the skin. A film forming agent can improvelong wear and non-transfer performance of a composition. Film formingagents can be water soluble, water insoluble, or water dispersing. Filmforming agents can be 1) organic silicone resins, fluorinated siliconeresins, copolymers of organic silicone resins, trimethylsiloxysilicate,GE's copolymers of silicone resins, SF1318 (silicone resin and anorganic ester of isostearic acid copolymer) and CF1301 (silicone resinand alpha methyl styrene copolymer), Dow Corning's pressure sensitiveadhesives copolymers of silicone resins and various PDMS's (BIO-PSAseries); and 2) acrylic and methacrylic polymers and resins,silicone-acrylate type copolymers and fluorinated versions of, includingsilicones plus polymer from 3M, KP545 from Shin-Etsu, alkyl-acrylatecopolymers, KP 561 and 562 from Shin-Etsu; 3) decene/butene copolymerfrom Collaborative Labs; 4) polyvinyl based materials, PVP, PVPNA,including Antaron/Ganex from ISP (PVP/Triacontene copolymer), Luviskolmaterials from BASF; polyurethanes, the Polyderm series from Alzoincluding but not limited to Polyderm PE/PA, Polyderm PPI-SI-WS,Polyderm PPI-GH, Luviset P.U.R. from BASF; 6) polyquaternium materials,Luviquat series from BASF; 7) acrylates copolymers andacrylates/acrylamide copolymers, Luvimer and Ultrahold series, bothavailable from BASF; 8) styrene based materials; and 9) chitosan andchitosan based materials including cellulose and cellulose-basedmaterials.

A composition can comprise a thickening agent or an emulsifying agent. Athickening agent may be used to increase the viscosity of liquid basematerials to be used in a cosmetic composition. The selection of aparticular thickening agent can depend on a type of composition desired(e.g., gel, cream, lotion, or wax based), the desired rheology, theliquid base material used, and other materials to be used in thecomposition. Examples of thickening agent or an emulsifying agent caninclude waxy materials such as candelilla, carnauba waxes, beeswax,spermaceti, carnauba, baysberry, montan, ozokerite, ceresin, paraffin,synthetic waxes such as Fisher-Tropsch waxes, silicone waxes (DC 2503from Dow Corning), microcrystalline waxes and the like; soaps, such asthe sodium and potassium salts of higher fatty acids, acids having from12 to 22 carbon atoms; amides of higher fatty acids; higher fatty acidamides of alkylolamines; dibenzaldehyde-monosorbitol acetals; alkalimetal and alkaline earth metal salts of the acetates, propionates andlactates; and mixtures thereof. Also useful are polymeric materials suchas, locust bean gum, sodium alginate, sodium caseinate, egg albumin,gelatin agar, carrageenin gum sodium alginate, xanthan gum, quince seedextract, tragacanth gum, starch, chemically modified starches and thelike, semi-synthetic polymeric materials such as cellulose, cellulosederivatives, cellulose ethers hydroxyethyl cellulose, methyl cellulose,hydroxypropyl cellulose, carboxymethyl cellulose, hydroxy propylmethylcellulose, polyvinylpyrrolidone, polyvinylalcohol, guar gum,hydroxypropyl guar gum, soluble starch, cationic celluloses, cationicguars and the like and synthetic polymeric materials such ascarboxyvinyl polymers, polyvinylpyrrolidone, polyvinyl alcoholpolyacrylic acid polymers, poly(acrylic acid), carbomers,polymethacrylic acid polymers, polyvinyl acetate polymers, polyvinylchloride polymers, polyvinylidene chloride polymers and the like.Inorganic thickeners may also be used such as aluminum silicates, suchas, for example, bentonites, or a mixture of polyethylene glycol andpolyethylene glycol stearate or distearate. An emulsifier may be used tohelp keep hydrophilic and hydrophobic ingredients from separating in anemulsion. In some cases, emulsifiers include but are not limited toOlivem, Oliwax LC, polysorbates, laureth-4, and potassium cetyl sulfate.

A cosmetic composition can provide a temporary change in an appearanceor can provide a long-term change in an appearance. In some cases, acosmetic composition can be formulated to provide a short-term change inan appearance (e.g., color deposition or plumping of skin) as well as along-term change in appearance (e.g., reduction in spots, appearance offine lines, appearance of wrinkles, or other features which can affectappearance).

A composition can comprise an additive that has an additive orsynergistic effect when applied with a polypeptide as disclosed herein.For example, a composition comprising a polypeptide and an additive canhave a greater effect on senescence, and age-related disease orcondition, or an age-associated disorder (e.g., delay the onset of,reduce the occurrence of, or ameliorate one or more symptoms) than theindividual effect of the additive, the polypeptide, or the sum of theindividual effects of the additive and the polypeptide. Additives can bean additional polypeptide, a glycosaminoglycan, a carbohydrate, apolyphenol, a protein, a lipid, a plant aqueous or oil extract, anucleic acid, an antibody, a small molecule, a vitamin, a humectant, anemollient, or another suitable additive. In some embodiments, thecomposition comprises a UV blocker. In some embodiments, the UV blockermay include but is not limited to aminobenzoic acid, avobenzone,cinoxate, dioxybenzone, homosalate, meradimate, octocrylene, octinoxate,octisalate, oxybenzone, padimate O, ensulizole, sulisobenzone, titaniumdioxide, trolamine salicylate, and zinc oxide.

Often the methods, systems, and compositions provided herein comprise avitamin. In some instances, the vitamin provides skin soothing, skinrestoring, skin replenishing, and/or hydrating effects. In someinstances, the vitamin provides antioxidant effects. In some instances,the vitamin acts as an emollient. In some instances, the vitaminimproves the appearance of enlarged pores, uneven skin tone, fine lines,dullness, and/or a weakened skin surface. In some instances, the vitaminis vitamin A, vitamin D, vitamin E, vitamin F, vitamin K, vitamin B1(thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5(pantothenic acid), vitamin B7 (biotin), vitamin B6, vitamin B12(cyanocobalamin), vitamin B9, folic acid, niacinamide, and mixturesthereof. In some instances, the composition comprises a derivative of avitamin. In some instances, a derivative of a vitamin is used to improvestability of the vitamin in the composition and/or compatibility of thevitamin derivative with other ingredients in the composition. In someinstances, the composition comprises vitamin B3 or its derivative andvitamin E or its derivative. In some instances, the compositioncomprises niacinamide and vitamin E or its derivative. In someinstances, the composition comprises vitamin C or its derivative,vitamin B3 or its derivative, and vitamin E or its derivative. In someembodiments, the composition comprises at least 0.01%, 0.05%, 0.5%, 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% vitamin. In some embodiments, thecomposition comprises about 0.1% to about 10%, about 0.1% to about 5%,about 0.5% to about 10%, about 0.5% to about 5%, about 1% to 10%, orabout 1% to 5% vitamin.

Compositions for topical administration can further comprise a carrier.The carrier may be a solution, an emulsion, an ointment, an oil, or agel base. The gel base, for example, may comprise one or more of thefollowing: petrolatum, lanolin, PEG(s), beeswax, mineral oil, diluentssuch as water and alcohol, emulsifiers, and/or stabilizers. Thickeningagents may be present in a therapeutic composition for topicaladministration. If intended for transdermal administration, thecomposition may include a transdermal patch or iontophoresis device. Insome cases, biodegradable microspheres (e.g., polylactic acid) may alsobe employed as carriers for a composition. In some cases, thetransdermal patch is prepared to deliver the formulation to theepidermal layer of the skin. In some cases, the transdermal patch isprepared to deliver the formulation to the epidermal and dermal layersof the skin. In some cases, the formulation is prepared as to beminimally delivered systemically in the subject or is not intended to bedelivered directly into the bloodstream of the subject.

A composition may also contain one or more diluents such as buffers, orone or more antioxidants such as ascorbic acid, low molecular weightpolypeptides, polypeptides, amino acids, carbohydrates includingglucose, sucrose or dextrins, chelating agents such as EDTA, glutathioneand other stabilizers. Neutral buffered saline or saline mixed withnonspecific serum albumin are exemplary diluents. A product may beformulated as a lyophilizate using suitable excipient solutions (e.g.,sucrose) as diluents.

A composition can also comprise one or more excipients, such as atherapeutic, nutraceutical, or cosmetic excipient. Examples ofexcipients can comprise antiadherents, binders, coatings, colors,disintegrants, flavors, glidants, lubricants, preservatives, sorbents,sweeteners, or vehicles.

Suitable excipients or stabilizers can be nontoxic to recipients at thedosages and concentrations employed, and can comprise buffers such asphosphate, citrate, and other organic acids; salts such as sodiumchloride; antioxidants including ascorbic acid, vitamin E, andmethionine; preservatives (such as, for example, octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride,benzethonium chloride; glucoonolactone and sodium benzoate; phenol,butyl or benzyl alcohol; low molecular weight (less than about 10residues) polypeptides; proteins such as serum albumin or gelatin;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA or EDTAalternatives (e.g. Biopure GLDA, Spectrastat G2); sugars such assucrose, mannitol, trehalose, or sorbitol; salt-forming counter-ionssuch as sodium; metal complexes (e.g., Zn-protein complexes); and/orsurfactants. In some instances, a surfactant includes, but is notlimited to, polysorbate 20, polysorbate 80, alginate, a poloxamer,TRITON (t-octylphcnoxypolyethoxyethanol); nonionic detergent; sodiumdodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside;lauryl-, myristyl-, linoleyl-, or stearyl-sulfohetame; lauryl-,yristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, orcetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroarnidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamin sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; sorbitan monopalmitate; and the MONAQUAT series(Mona Industries. Inc., Paterson, N.J.); polyethylene glycol (PEG),polypropylene glycol (PPG), copolymers of poloxyethylene andpoloxypropylene glycol (e.g., Pluronies/Poloxamer, PLURONIC® F68, etc.);or another suitable surfactant. In some cases, the composition cancomprise squalene, natural oils, plant extracts, hyaluronic acid, orclay. In some cases, the composition comprises a skin penetratingenhancer to enhance the penetration of active ingredients into the skin.In some cases, the skin penetrating enhancer may include but are notlimited to fatty acids, essential oils, urea, liposomes, microsphere,DMSO, azone, sodium PCA, and squalene.

In some embodiments, the formulation comprises a carrier, a microsphere,a liposome, or a micelle in order to carry the polypeptide and controlthe release time and/or penetration depth of the polypeptide in thethrough the skin.

In some embodiments, the polypeptide is functionalized. In someembodiments, the polypeptide is functionalized with a chemical group. Insome embodiments, the polypeptide is functionalized with a functionalgroup comprising no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or more than 20 carbons. In some embodiments,the polypeptide is functionalized with acetyl or palmitoyl.

A polypeptide or composition to be applied to a subject can besterilized. This may be accomplished by, for example, filtration throughsterile filtration membranes, or any other art-recognized method forsterilization.

A composition can comprise a therapeutically effective amount of apolypeptide or peptidomimetic in an amount which can delay the onset of,reduce the occurrence of, or ameliorate one or more symptoms, such as asymptom of the skin, of an age-related disease or condition, orage-associated disorder. In some cases, a therapeutically effectiveamount can be an amount of a therapeutic agent (e.g., a polypeptide)that can provoke a therapeutic (e.g., senotherapeutic) or desiredresponse in a subject. A therapeutically effective amount can besufficient to cause a therapeutic benefit to the subject. Thetherapeutically effective amount can vary depending on a variety offactors including the active agent selected for use, and the age,weight, height, and/or general health of a subject to be treated.

As is understood in the clinical context, an effective therapeuticamount of an active agent may or may not be achieved in conjunction withanother drug, compound, therapeutic, or pharmaceutical composition.Thus, an effective therapeutic amount may be considered in the contextof administering one or more active agents, and a single active agentmay be considered to be given in an effective amount if, in conjunctionwith one or more other active agents, a desirable result may be or isachieved. Accordingly, in some instances, one or more active agents maybe administered to the subject. In other instances, treatment with anactive agent described herein is conducted prior to, or after, one ormore other treatment modalities described herein.

Polypeptide Synthesis

Also disclosed are isolated polynucleotides encoding one or more of thepresently disclosed polypeptides. The isolated polynucleotides may bepresent in an expression vector comprising the isolated polynucleotidesoperably linked to a promoter. The expression vector may be present inan isolated cell (i.e., a recombinant cell transfected or transformedwith the expression vector).

Suitable expression vectors may include bacterial, plant, fungal,insect, or animal host cell replication, and/or expression vectors thatexpress the disclosed peptides, polypeptides, and variants thereof.Expression vectors may be used to transform appropriate host cells(e.g., E. coli). The transformed host cell may be cultivated orfermented such that the peptide or polypeptide is expressedconstitutively or after adding a reagent that induces expression (e.g.,via an inducible promoter). Expression vectors as contemplated hereinmay include control sequences that modulate expression of the encodedpolypeptide. Expression control sequences may include constitutive orinducible promoters (e.g., T3, T7, Lac, trp, or phoA), ribosome bindingsites, or transcription terminators.

An expression vector can be utilized to transform host cells. Suitablehost cells include bacterial, plant, fungal, insect, or animal hostcells. Suitable bacteria include, but are not limited to: Gram-negativebacteria such as Escherichia species (e.g., E. coli), otherGram-negative bacteria, (e.g., Pseudomonas sp., such as Pseudomonasaeruginosa, or Caulobacter sp., such as Caulobacter crescentus), orGram-positive bacteria (e.g., Bacillus sp., such as Bacillus subtilis).Suitable fungal cells may include yeast (e.g., Saccharomycescerevisiae).

An expression vector can, for example, provide a mechanism for synthesisof a polypeptide. Synthesis can take place in a cell, for example ananimal cell, a plant cell, a bacterial cell, or a yeast cell. Anexpression vector can comprise nucleic acids, e.g., DNA derived from aplasmid, cosmid, phasmid, or bacteriophage or synthesized by chemical orenzymatic means, into which one or more fragments of nucleic acid may beinserted or cloned which can encode one or more polypeptides describedherein. An expression vector may be capable of autonomous replication ina defined host or organism such that the cloned sequence is reproduced.An expression vector can have a linear, circular, or supercoiledconfiguration and may be complexed with other vectors or other materialsfor certain purposes. The components of an expression vector cancomprise, but are not limited to, a DNA molecule incorporating: (1) DNA;(2) a sequence encoding a therapeutic or desired product; or (3)regulatory elements for transcription, translation, RNA stability, andreplication.

A polypeptide can be produced using an expression vector. In some cases,such production can comprise cultivating or fermenting a transformedhost cell (e.g., a bacterial host cell as contemplated herein) whichcomprises an expression vector (as contemplated herein) which in turncomprises a nucleic acid molecule encoding the disclosed peptides,polypeptides, or variants thereof (as contemplated herein), whereincultivation occurs under conditions which cause expression of thepeptides, polypeptides, or variants; and isolating, separating, orpurifying the peptides, polypeptides, or variants. The transformedbacteria may be cultivated or fermented using methods known in the artin order to express the peptides, polypeptides, or variants. Anexemplary isolation, separation, or purification method may include oneor more of the following steps: a cell disruption step, a clarificationstep (e.g., via centrifugation or filtration), a chromatographicseparation step, a dialysis step, and a precipitation step.

In some other embodiments, the polypeptide can be chemicallysynthesized. Synthesis of a polypeptide can be performed usingsolution-phase techniques, solid-phase methods, or other suitablemethods of polypeptide synthesis.

Methods

Provided are methods for the use of the polypeptides and compositionsdisclosed herein. Such methods can comprise application of one or moreof the polypeptides described herein to a subject. Methods describedherein can delay the onset of, reduce the occurrence of, or amelioratean age-related disease or condition or age-associated disorder.

Methods described herein can delay the onset of, reduce the occurrenceof, reduce the appearance of, or ameliorate a disease, a disorder, or acondition associated with the accumulation of senescent cells. A diseaseor disorder associated with the accumulation of senescent cells can beage related. In some cases, the disease or disorder can worsen over timeif untreated.

A polypeptide or composition can be applied or administered to a subjectto treat a condition directly or indirectly influenced by skin health.Such a method can comprise administering to the subject a compound thatpromotes skin health or applying topical treatment to the skin.

An age-associated disorder can comprise a biological progression ofevents that occurs during a disease process that can affect the body,which can mimic or substantially mimic all or part of the aging eventswhich occur in a normal subject. In some cases, this biologicalprogression of events can occur over an accelerated time frame.

An age-related disease or condition or age-associated disorder canrelate to regular processes in the body, such as movement and eatingcapacity.

In some cases, the age-related disease or condition or age-associateddisorder can be a disease, condition, or disorder affecting the skin,such as a skin disorder or a dermatosis, which can comprise wrinkles,lines, dryness, itchiness, spots, age spots, bedsores, ulcers, cancer,dyspigmentation, infection (e.g., fungal infection), or a reduction in askin property such as clarity, texture, elasticity, color, tone,pliability, firmness, tightness, smoothness, thickness, radiance,luminescence, hydration, water retention, skin barrier, evenness,laxity, or oiliness, or other dermatoses. In some instances, theage-related disease or condition or age-associated disorder ishyperpigmentation of the skin. In some instances, the hyperpigmentationdisorder is melasma, age spots, lentigines, and/or progressivepigmentary purpura. In some instances, the hyperpigmentation is a resultof sun damage, inflammation, hormone changes, or skin injuries. In someinstances, the hyperpigmentation occurs after a cosmetic procedure,including but not limited to a laser treatment, a light treatment, or achemical peel; administration of an antibiotic, an oral contraceptive,or a photosensitizing drug; or application of a topical agent. In someinstances, the hyperpigmentation is a result of excess production ofmelanin.

In some instances, treatment of the age-related disease or condition orage-associated disorder with the methods, systems and compositionsdisclosed herein results in lightening, increasing luminescence,brightening, evening, smoothing and/or firming of the skin's appearance.In some instances, treatment with the methods, systems, and compositionsdisclosed herein improves the epidermal barrier, skin hydration level,skin water retention, appearance of wrinkles, smoothness, firmness,elasticity, appearance of radiance and luminosity, and/or improves ormaintains the ceramide level in the skin. In some instances, the effectof treatment with the methods, systems, and compositions disclosedherein is assessed by measuring skin moisture content, trans-epidermalwater loss (TEWL), dermal thickness and echogenicity, intracutaneousanalysis, skin viscoelastic properties, or skin surface profile. In someinstances, the effect of treatment with the methods, systems, andcompositions disclosed herein assesses for reduction in appearance oflines/wrinkles, appearance of skin tone (evenness), appearance of pores,appearance of texture/smoothness, firmness (visual), elasticity(tactile), epidermal barrier, skin roughness, skin hyperpigmentation, oroverall appearance. In some instances, the effect of treatment with themethods, systems, and compositions disclosed herein is measured using aninstrument, including but not limited to a corneometer for measuringskin moisture content /hydration, a VapoMeter for measuring thetrans-epidermal water loss (TEWL), an ultrasound measuring dermalthickness (density) and echogenicity, a non-invasive optical skinimaging instrument for measuring skin evenness and chromophore mapping,a cutometer using suction for measuring viscoelastic properties of theskin (firmness and elasticity), skin profilometry, multi-spectralanalysis, and colorimetry for measuring skin surface profile, lines, andwrinkles

In some instances, the treatment of the age-related disease or conditionor age-associated disorder results in the reduction of the appearance ofwrinkles or skin pigmentation by, for example, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 99%. In some instances, the instrumentalmeasurement shows an improvement in at least one of appearance oflines/wrinkles, appearance of skin tone (evenness), appearance of pores,appearance of texture/smoothness, firmness (visual), elasticity(tactile), epidermal barrier, skin roughness, skin radiance, or overallappearance by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% after the use of thecomposition as compared to before the use of the composition. In someinstances, the improvement is presented as a mean percentage improvement(MPI) compared to baseline before the use of the composition. In someinstances, MPI is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% after the use of thecomposition as compared to before the use of the composition. In someinstances, the measurements are taken at 1 week, 2 weeks, 3 weeks, 4weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks,12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 5 months, 6 months, orone year after the use of the composition. In some instances, the effectof the treatment is assessed by an expert in skin conditions, disorders,or diseases who analyzes one or more of the skin measurements. In someinstances, the effect of the treatment is assessed by the userthemselves. In some instances, at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% of the users may report an improvement in at least oneof skin barrier, skin roughness, skin radiance, appearance oflines/wrinkles, appearance of skin tone (evenness), appearance of pores,appearance of texture/smoothness, firmness (visual), elasticity(tactile), or overall appearance after using the methods, systems, andcompositions disclosed herein. In some instances, at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% of the users may report animprovement in hydration of the skin. In some instances, at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the users may report animprovement in skin barrier function.

In some instances, the methods, systems, and compositions providedherein may reduce hyperpigmentation of the skin. In some instances,hyperpigmentation is associated with excess production of melanin. Insome instances, the methods, systems, and compositions provided hereinreduces the excess production of melanin. In some instances, themethods, systems, and compositions provided herein reduce the presenceof melanin pigment in the skin. In some instances, the methods, systems,and compositions provided herein reduce the expression levels ofproteins involved in melanogenesis, including tyrosinase, melanocyteinducing transcription factor (MITF) and dopachrome tautomerase (DCT),by the cells in the treated skin. In some instances, the methods,systems, and compositions provided herein result in reduction oftyrosinase activity, reduction of the expression or activation oftyrosinase, scavenging of the intermediate products of melaninsynthesis, reducing the transfer of melanosomes to keratinocytes,reduction of existing melanin content, or reduction in melanocyteactivity or viability.

In some cases, the methods, systems, and compositions provided hereinmay reduce cutaneous inflammation. In some cases, the methods, systems,and compositions provided herein may reduce expression levels ofproteins involved in inflammation, interferon-gamma (IFN-γ) andinterleukin 10 (IL-10), by the cells in the treated skin.

In some embodiments, the compositions described herein are administeredonce per day, twice per day, three times per day or more. In someembodiments, the compositions described herein are administered twicedaily administration, e.g., morning and evening. In some embodiments,the compositions described herein are administered daily, every day,every alternate day, five days a week, once a week, every other week,two weeks per month, three weeks per month, once a month, twice a month,three times per month, or more. In some embodiments, the compositionsdescribed herein are administered for at least 1 week, 2 weeks, 3 weeks,1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3years, 4 years, 5 years, or longer. In some embodiments, the compositionis directed to be applied as a smooth layer onto clean, dry skin on faceand/or neck in the morning and the evening. In some embodiments,formulation is a daily essential topical supplement scientificallyformulated to improve skin resilience and strengthen epidermal barrierfor long-lasting health of the skin. In some embodiments, the userapplies the compositions described herein comprising at least one of thepolypeptides to the face and/or neck. In some embodiments, thecomposition is directed to be applied to the skin on the body. In someembodiments, the compositions described herein is used in conjunctionwith other topical compositions, such as a UV blocker. In someembodiments, the compositions described herein is applied before,together with, or after the application of the other topicalcomposition. In some embodiments, the composition comprises a UVblocker.

A polypeptide or composition can be applied topically, i.e., to skin, todelay the onset of, reduce the occurrence of, or ameliorate the disease,condition, or disorder affecting the skin.

An age-related disease or condition or age-associated disorder can becaused by UV damage, DNA damage, ATRX foci accumulation in cell nuclei,increased p16 expression, increased senescence-associatedβ-galactosidase activity, accumulation of senescent cells in the tissue,increased SASP production, chemically induced senescence, chronologicalaging, decreased hyaluronic acid production, decreased expression ofsirtuin 6, altered insulin-like growth factor-1 (IGF-I) pathwaysignaling, increased production of matrix metallopeptidase 1 (MMP1),thin epidermal layer of the skin, or genetic variants. In someinstances, the age-related disease or condition or age-associateddisorder is initiated or exacerbated by a therapeutic regimen, forexample, a side effect of a therapeutic drug. An age-related disease orcondition or age-associated disorder can affect the health or appearanceof skin directly or indirectly. Topical application of a polypeptide orcomposition herein can improve the health or appearance of skin in somesuch cases.

An age-related disease or condition or age-associated disorder cancomprise a cell proliferative disorder. A cell-proliferative disordercan affect the health or appearance of the skin. In some cases, atreatment administered for a cell-proliferative disorder, such aschemotherapy or radiation can affect the health or appearance of theskin. Topical application of a polypeptide or composition herein canimprove the health or appearance of skin in some such cases.

Also provided herein are methods for treating the skin of a subjectcomprising administering to a subject a composition that can promote adecrease in a number of senescent cells in a tissue or organism,inducing a pro-apoptotic state in the treated cells, inducing SIRT6expression, preventing DNA-induced senescence, and/or enhancing DNArepair capacity. In some cases, a skin disease such as a dermatologicaldisease or condition can comprise skin sagging or wrinkling,accumulation of senescent cells in the tissue, decreased epidermalthickness, decreased collagen production, increased MMP-1 production,decreased DNA repair capacity, decreased SIRT6 expression, skindisorganization, a thin epidermal layer of the skin, inflammation, asenescence-associated secretory phenotype, or stem cell exhaustion ofthe skin.

Methods can comprise administering to the subject a compositioncomprising a polypeptide that can promote a significant decrease in thenumber of senescent cells in the tissue or organism. A decrease in thenumber of senescent cells can comprise a pro-apoptotic state in thetreated cells, inducing SIRT6 expression, preventing DNA-inducedsenescence, or enhancing DNA repair capacity. In some cases, the numberof senescent cells in a sample, a portion of a subject (e.g., the facialskin of a subject), and/or a subject can be reduced by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, or at least 90%.

A polypeptide or compositions can be applied or administered to a cell,tissue, or subject. In some cases, application or administration of apolypeptide can result in a senotherapeutic effect in a cell, tissue, orsubject. In some cases, a polypeptide can be administered to a subject,applied topically to a subject, or incubated with cultured cells toprovide a senotherapeutic effect.

A cell can be a cultured cell or a cell isolated from a subject or froma cell line. Some examples of cultured cells can comprise a keratinocyteor a fibroblast or a melanocyte. A cell can be wild type or can begenetically modified. Some genetic modifications can promote senescence,such as genetic modifications in the p53/p21 pathway, the p16/RBpathway, the, a mRNA or miR gene, among other RNA classes. In somecases, application of a polypeptide or composition to a cell can reducesenescence in the cell. In some cases, cells may comprise cells in vivoor in situ in an organism, including but not limited to animals, C.elegans, and humans.

A tissue can be a tissue which is a tissue of a subject or a tissuewhich has been isolated from a subject, i.e., ex vivo. In some cases, atissue can be artificially grown. A tissue can comprise skin, andexamples of tissues can comprise healthy skin, diseased skin, aged skin,or scalp. In some cases, application of a polypeptide or composition toa tissue can reduce senescence in one or more cells of the tissue or theentire tissue. In some cases, tissue may comprise tissue in vivo or insitu in an organism, including but not limited to animals, C. elegans,and humans.

In cases where the subject is a human, the subject may be of any age. Insome cases, the subject has an age-related disease or condition orage-associated disorder, is at risk for an age-related disease orcondition or age-associated disorder, or is healthy. A subject can bemale or female.

A method can comprise topical application of a polypeptide orcomposition. Topical application can comprise rubbing, spraying,dipping, dabbing, or otherwise applying a polypeptide or composition toskin or mucosa.

EXAMPLES Example 1

Primary fibroblasts isolated from Progeria patients can constitute agenetic model of early aging and cellular senescence in humans. Primaryfibroblasts from Progeria patients were cultured in DMEM (Dulbecco'sModified Eagle's Medium) supplemented with 10% v.v. fetal bovine serum(FBS), and 1% v.v. of penicillin/streptomycin solution (1,000 U·mL-1).Cells were kept in a 5% CO₂, 37° C. and 95% humidity atmosphere. Afterexpansion, these cells were seeded in 96-well plates (1,000 cells perwell) and, 6 hours after plating, were incubated with individualpolypeptides from a proprietary library at 50 μM for 48 hours. Negativecontrol comprised untreated cells, which received vehicle only; positivecontrol group comprised cells incubated with 10 μM ABT-263, a senolyticcompound for the same period. After incubation, relative cellularsenescence (assessed by the activity of senescence associatedB-galactosidase staining relative to untreated control) was analyzed asshown in FIG. 1, in which the Y axis indicates the total number of cellsin the well (normalized to the untreated control), and the X axisrepresents senescence associated B-galactosidase stainingintensity/nuclei (i.e., senescence level), also normalized to negativecontrol. Three independent experiments including three technicalreplicates were performed. Polypeptides which promoted a significantdecrease of cellular senescence to below 75% that of the untreatedcontrol sample were considered positive hits.

A total of 764 polypeptides were tested, among which 56 promoted adecrease in cellular senescence to below 75% that of the untreatedcontrol sample. Therefore, they were considered as positive hits, andputative senotherapeutic compounds. ABT-263, which was considered apositive control in the experiment, also promoted significant reductionof cellular senescence, but also cellular toxicity. This observationconfirmed the senolytic characteristic of ABT-263, as well as thesenotherapeutic potential of some of the tested polypeptides (FIG. 1).

Example 2

Primary fibroblasts isolated from 3 healthy chronologically agedpatients were used. Cells were cultured in DMEM (Dulbecco's ModifiedEagle's Medium) supplemented with 10% v.v. fetal bovine serum (FBS), and1% v.v. of penicillin/streptomycin solution (1,000 U·mL-1). Cells werekept in a 5% CO₂, 37° C. and 95% humidity atmosphere. After expansion,these cells were seeded in 96-well plates (4,000 cells per well) and, 6hours after plating the cells were treated with one of 4 senotherapeuticpolypeptides (Peptide 14, Peptide 13, Peptide 15, and Peptide 16) andincubated for 48 hours. Each polypeptide was tested at 6 differentconcentrations: 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.12 μM, and 1.56 μM,except for Peptide 16, which was tested at 5 different concentrations:25 μM, 12.5 μM, 6.25 μM, 3.12 μM, and 1.56 μM. A negative controlcomprised untreated cells, which received vehicle only. Afterincubation, relative cellular senescence (assessed by the activity ofsenescence associated B-galactosidase staining relative to untreatedcontrol) was analyzed (FIG. 2, panels A-D), in which the Y axisindicates the relative senescence levels normalized to untreatedcontrol. Each column corresponds to a different concentration of apolypeptide. Three independent experiments (biological replicates)including three technical replicates were performed. Data was analyzedusing Analysis of Variance (ANOVA) and a Bonferroni post-hoc tests.Statistical significance was determined as p values equal or lower than0.05.

All polypeptides presented senotherapeutic potential in at least one ofthe tested concentrations, evidenced by the significant reduction ofcellular senescence compared to untreated control. *p<0.05; **p<0.01;***p<0.001 compared to untreated control (ctrl) (FIG. 2, panels A-D).

Example 3

ATRX is a chromatin remodeling enzyme, which contributes to theformation of senescence associated heterochromatic foci. It increasinglyaccumulates in nuclear foci during senescence. Therefore, it constitutesa marker of cellular senescence. To investigate whether Peptide 14decreased levels of cellular senescence, ATRX foci were analyzed inPeptide 14 treated (1 μM, 500 nM, 100 nM, and 10 nM) and untreatedcells. To do so, primary fibroblasts isolated from 3 healthychronologically aged (elder) donors were used. These cells were culturedin DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% v.v.fetal bovine serum (FBS) and 1% v.v. of penicillin/streptomycin solution(1,000 U·mL-1). Cells were kept in a 5% CO₂, 37° C. and 95% humidityatmosphere. After expansion, these cells were seeded in 96-well plates(4,000 cells per well) and, 6 hours after plating, were incubated for 48hours with the Peptide 14 polypeptide in the aforementionedconcentrations. A negative control comprised untreated cells, whichreceived vehicle only. After incubation, relative cellular senescencewas assessed. Briefly, immunostaining was performed by fixing,permeabilizing, and incubating the cells with anti-ATRX antibody,followed by secondary antibody. The number of nuclei and stained ATRXfoci were counted. Panel A of FIG. 3 shows a representative graphshowing the number of cells (Y axis) presenting a specific amount ofATRX foci/cell, represented as columns (X axis). The upper graph depictsuntreated cells, while the lower graph depicts cells treated withPeptide 14 at 500 nM. Panel B of FIG. 3 shows the average number of ATRXfoci/nuclei of fibroblasts treated with different conditions of Peptide14 (columns). Panel C of FIG. 3 shows the percentage of cells presentingless than 10 ATRX foci/nuclei among fibroblasts treated with differentconditions of Peptide 14 (columns). Three independent experiments(biological replicates) including three technical replicates wereperformed. Data in panels B and C of FIG. 3 were analyzed using ANOVAand Bonferroni post-hoc tests. Statistical significance was determinedas p values equal or lower than 0.05.

Peptide 14 treatment significantly decreased ATRX foci/nuclei, when usedat 500 nM and 50 nM, compared to untreated cells. In the sameconcentrations, Peptide 14 also increased the number of cells whichpresented less than 10 foci/nuclei. *p<0.05; **p<0.01 compared tountreated control (ctrl) (FIG. 3).

Example 4

Human primary fibroblasts isolated from 3 healthy chronologically aged(elder) donors were used. These cells were cultured in DMEM (Dulbecco'sModified Eagle's Medium) supplemented with 10% v.v. fetal bovine serum(FBS), and 1% v.v. of penicillin/streptomycin solution (1,000 U·mL-1).Cells were kept in a 5% CO₂, 37° C. and 95% humidity atmosphere. Afterexpansion, these cells were seeded in T-75 flasks (250,000 cells perflask) and, 6 hours after plating, were incubated for 3 weeks (21 days)with Peptide 14 at 3.12 μM. A negative control comprised untreatedcells, which received vehicle only. Day 0 was defined as the day cellsstarted to be treated with Peptide 14. No Peptide 14 treatment wasperformed between days 21 and 28. Weekly, cellular senescence wasassessed according to senescence associated β-galactosidase staininglevels. Data was normalized to untreated group and plotted (FIG. 4,panel A). Cellular proliferation was also determined weekly. At days 7,14, 21, and 28, cells were trypsinized and counted (FIG. 4, panel B).After counting, 250,000 cells were plated in new T-75 flasks. Threeindependent experiments (biological replicates) including threetechnical replicates were performed. Data was analyzed using T-test.Statistical significance was determined as p values equal or lower than0.05.

Peptide 14 promoted a significant decrease (***p<0.001; ****p<0.0001) ofcellular senescence beginning at the second week. After 21 days oftreatment, the senotherapeutic effect of Peptide 14 was maintained forat least 7 days after polypeptide removal (between experimental days 21and 28). No significant differences were observed regarding cellproliferation comparing Peptide 14 treated group and untreated control(FIG. 4).

Example 5

Primary fibroblasts isolated from 7 healthy chronologically agedpatients were used (patients were aleatory identified as patient 2, 3,4, 5, 6, 7, or 8). These cells were cultured in DMEM (Dulbecco'sModified Eagle's Medium) supplemented with 10% v.v. fetal bovine serum(FBS), and 1% v.v. of penicillin/streptomycin solution (1,000 U·mL-1).Cells were kept in a 5% CO₂, 37° C. and 95% humidity atmosphere. Afterexpansion, these cells were seeded in 96-well plates (4,000 cells perwell) and, 6 hours after plating, were incubated for 48 hours withPeptide 14 at 5 different concentrations: 25 μM (concentration 5), 12.5μM (concentration 4), 6.25 μM (concentration 3), 3.12 μM (concentration2), and 1.56 μM (concentration 1). A negative control compriseduntreated cells (concentration 0), which received vehicle only. Afterincubation, relative cellular senescence was determined according to theaverage number of ATRX foci/nuclei quantified following ATRXimmunostaining. Seven independent experiments (biological replicates)including three technical replicates were performed. Data was analyzedusing a covariance test. Statistical significance was determined asp-values equal or lower than 0.05 (FIG. 5).

Covariance analysis shows that the number of ATRX foci/nuclei wassignificantly reduced following Peptide 14 treatment. Peptide 14efficacy followed a dose-response pattern, with concentration and ATRXfoci/nuclei being significantly correlated (p<0.0004) (FIG. 5).

Example 6

Cellular senescence may be caused by several different stimuli. In orderto assess whether Peptide 14 was effective against UVB-induced andchemically-induced senescence, human primary fibroblasts isolated from 3healthy donors were used. These cells were cultured in DMEM (Dulbecco'sModified Eagle's Medium) supplemented with 10% v.v. fetal bovine serum(FBS), and 1% v.v. of penicillin/streptomycin solution (1,000 U·mL-1).Cells were kept in a 5% CO₂, 37° C. and 95% humidity atmosphere. Afterexpansion, these cells were seeded in 96-well plates (4,000 cells perwell) and, 6 hours after plating, were submitted to either etoposide (20μM) treatment for 24 hours, or exposed twice to 0.05 J/cm² of UVBradiation. Each UVB exposure corresponds to approximately 1 to 3 hoursof daily sun exposure in April of major cities around the world (e.g.Auckland, NZ; Los Angeles, US; and Brasilia, BR). After the differentsenescence induction protocols, etoposide treated cells were incubatedwith Peptide 14 at 5 μM, 2.5 μM, or 1 μM for 48 hours. UVB exposed cellswere treated with Peptide 14 at 5 μM for 48 hours. A negative controlcomprised untreated cells, which were submitted to stress, but receivedvehicle only. After incubation, relative cellular senescence (assessedby the activity of senescence associated β-galactosidase stainingrelative to untreated control) was analyzed and plotted in columngraphs. ATRX foci were also assessed following ATRX immunofluorescencestaining. Graphs were built using the average ATRX foci detected pernuclei. Three independent experiments (biological replicates) includingthree technical replicates were performed. Data was analyzed using witheither t-test or ANOVA followed by a Bonferroni post-hoc test.Statistical significance was determined as p-values equal or lower than0.05 (FIG. 6).

Etoposide treatment promoted a significant increase in cellularsenescence level (p<0.001), and also a significant increase of ATRX focinuclear accumulation (represented as the average number of ATRXfoci/cell; p<0.05) (FIG. 6, panel A). When etoposide-stressed cells weretreated with 2.5 μM or 5 μM of Peptide 14, senescence associatedβ-galactosidase staining was significantly reduced (*p<0.05) (FIG. 6,panel B, left graph). Average ATRX foci/cell was also significantlyreduced when etoposide-exposed cells were treated with 2.5 μM Peptide14, as shown in the right graph of panel B of FIG. 6. UVB exposure alsopromoted significant increase in cellular senescence, as assessed bysenescence associated β-Galactosidase staining, 5 μM Peptide 14treatment being able to significantly prevent cellular senescence(*p<0.05), as shown in the left graph of panel C of FIG. 6. Treatmentwith Peptide 14 did not significantly alter cell number, as shown in theright graph of panel C of FIG. 6. UVB exposure promoted significantincrease in the average number of ATRX foci per nuclei, and 5 μM Peptide14 treatment significantly prevented cellular senescence, leading tosignificantly reduced ATRX foci/nuclei, compared to UVB treated sampleswhich did not receive Peptide 14 (*p<0.01) (FIG. 6, panel D).

Example 7

Human primary fibroblasts and keratinocytes isolated from healthy elderdonors were used to build human skin equivalents. Those skin equivalentswere treated with 0.01% w.v. Peptide 14 for 5 days and werecharacterized according to levels of senescence using senescenceassociated B-galactosidase staining, overall structure, as indicated byepidermal thickness. Quality assessment based several parameters wasperformed by blind analysts. The observed parameters include generalorganization of cell layers, as well as the thickness of the hornylayer, among other aspects and were shown to decrease with aging andsenescence level. The assessment had a maximum score of 28, where higherscore correlated with decrease in age and senescence. A minimal score of19 was required for batch use. This score was validated internally andshown to decrease with age/senescence of the skin equivalents orcultured cells. Furthermore, skin equivalents were characterizedaccording to the expression of specific genes by reversetranscription-quantitative polymerase chain Reaction (RT-qPCR).Following treatment, epidermis and dermis were processed for RT-qPCRseparately. For epidermis samples, glyceraldehyde 3-phosphatedehydrogenase (GAPDH; ubiquitously expressed); p16 (associated tosenescence), IL-8 (linked to irritation), and Ki-67 (associated to cellproliferation) were analyzed. For dermis samples, glyceraldehyde3-phosphate dehydrogenase (GAPDH; ubiquitously expressed); p16(associated to senescence), IL-8 (linked to irritation), Ki-67(associated to cell proliferation); hyaluronic synthase 2 (HAS-2;associated with hyaluronic acid production), and matrix metalloprotease1 (MMP1; associated to extracellular matrix protein degradation) wereanalyzed. CT values were analyzed using the 2^(−ΔΔct) method. AveragemRNA expression was normalized to GAPDH (ΔCt) and to the negativecontrol group (ΔΔCt). Negative controls received formulation only. Threeindependent experiments were performed with three technical replicates.Data was analyzed using T-test. Statistical significance was determinedas p-values equal or lower than 0.05 (FIG. 7)

Peptide 14 treatment of human skin equivalents promoted increased skinequivalent score (Peptide 14 group 24±1 versus 19.0±2), as shown inpanel A of FIG. 7, suggesting treatment safety, tolerability andbeneficial effects. This is in contrast to other senolytic agents thatmay have safety and tolerability issues. See, e.g., Tse et al., CancerRes. 68: 3421 (2008); Wilson et al., Lancet Oncol. 11:1149 (2010). Inaddition, treatment promoted a significant decrease in senescenceassociated β-galactosidase staining (***p<0.001), as shown in panel A ofFIG. 7 and panel B of FIG. 7, corroborating the senotherapeutic effectof the polypeptide. Furthermore, Peptide 14 treatment led to significantdecrease of p16 in epidermis (****p<0.0001) and dermis (**p<0.01);decreased expression of IL-8 in the dermis (*p<0.5); and decreasedexpression MMP-1 in the dermis (**p<0.01). The data corroborates thesenotherapeutic potential of Peptide 14, safety and tolerability, aswell as the beneficial effects of the polypeptide to skin geneexpression, as shown in panel C of FIG. 7.

Example 8

In order to shed light into the mechanism of action of Peptide 14 andsimilar Peptide 13, Akt 5473 phosphorylation (FIG. 8, panel A),senescence associated β-galactosidase staining (FIG. 8, panel B), aswell as mRNA expression (FIG. 8, panel C) were investigated. For AktS473 phosphorylation analysis using western blotting, human primaryfibroblasts and keratinocytes were used. These cells were used to buildhuman skin equivalents, which were kept in a 5% CO₂, 37° C. and 95%humidity atmosphere air liquid interface. Then, skin equivalents weretreated with 1 μM of either Peptide 14 or Peptide 13 for 5 days, andprotein analysis was performed on the skin equivalents. Protein wasisolated and quantified. Equal amounts of protein were loaded inpolyacrylamide gels and transferred into a nitrocellulose membrane.GAPDH (loading control) and pAkt S473 antibodies were incubated with themembrane and staining was revealed by chemiluminescence. Relative pAkt5473/GAPDH signal was compared between treated and non-treated samples.For senescence associated B-galactosidase staining experiments,fibroblasts were used. These cells were cultured in DMEM (Dulbecco'sModified Eagle's Medium) culture supplemented with 10% v.v. fetal bovineserum (FBS), and 1% v.v. of penicillin/streptomycin solution (1,000U·mL-1). Cells were kept in a 5% CO₂, 37° C. and 95% humidityatmosphere. After expansion, these cells were seeded in 96-well plates(4,000 cells per well) and incubated with basal medium for 6 hours toallow cell attachment. After, cells were exposed twice to 0.05 J/cm².This was followed immediately by a second incubation, wherein Peptide 14or Peptide 13 were added to the medium and left for 48 hours, whenmedium was changed, and cells were stained for senescence associatedB-galactosidase. Untreated cells were incubated with vehicle only asnegative controls (−). Relative staining was obtained after normalizinguntreated control senescence associated B-galactosidase levels to 100%.For mRNA analysis, fibroblasts were used. These cells were cultured inDMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% v.v.fetal bovine serum (FBS), and 1% v.v. of penicillin/streptomycinsolution (1,000 U·mL-1). Cells were kept in a 5% CO₂, 37° C. and 95%humidity atmosphere. After expansion, these cells were seeded in either6-well plates (50,000 cells per well) and incubated with basal mediumfor 6 hours to allow cell attachment. After, cells were incubated for 48hours with either Peptide 14 or Peptide 13. A negative control compriseduntreated cells, which received vehicle only. Total RNA was isolated,samples were reverse transcribed, and mRNA expression of GAPDH, sirtuin6 (SIRT6), BLM, and exonuclease 1 (EXO1) genes were determined usingqPCR. Negative controls received vehicle only. CT values were analyzedusing the 2^(−ΔΔct) method. Average mRNA expression was normalized toGAPDH (ΔCt) and to the negative control group (ΔΔCt). For all analysis,three independent experiments were performed with three technicalreplicates. Data was analyzed using a T-test. Statistical significancewas determined as p values equal or lower than 0.05 (FIG. 8).

pAkt 5473 was significantly decreased in both epidermal, as well asdermal samples treated with Peptide 14 (*p<0.05, and **p<0.01,respectively). Peptide 13 decreased pAkt S473 in dermal samples only(***p<0.001) (FIG. 8, panel A). For UVB and senescence associatedB-galactosidase staining, it was observed that staining always increasedfollowing UVB exposure. Additionally, both Peptide 14 and Peptide 13decreased staining in UVB-exposed samples (***p<0.001). Peptide 14specifically led to increased SIRT6 and BLM expression in treatedsamples (*p<0.05) (FIG. 8).

Example 9

Human primary fibroblasts and keratinocytes isolated from healthy elderdonors were used to build human skin equivalents. Those skin equivalentswere treated with 0.01% w.v. of Peptide 14 for 5 days and characterizedaccording to epidermal thickness, which was quantified according tototal epidermal area. Negative controls were treated with formulationonly. Example histological images are shown in panel A of FIG. 9. Threeindependent experiments were performed with three technical replicates.Data was analyzed using a t-test. Statistical significance wasdetermined as p-values equal or lower than 0.05 (panel B of FIG. 9).

Peptide 14 treatment of human skin equivalents promoted increasedepidermal thickness (**p<0.01), compared to untreated control,suggesting beneficial effects of the polypeptide over skin epidermis(FIG. 9).

Example 10

Predicted three-dimensional structures of polypeptides having the aminoacid sequence ETAKHWLKGI (SEQ ID NO:1) and ATAKAWLKGI (SEQ ID NO:2) weredetermined in water. The structure predictions are shown in FIG. 10,panel A (SEQ ID NO:1) and FIG. 10, panel B (SEQ ID NO:2). The structureswere superimposed (FIG. 10, panel C) to illustrate the resemblance ofthe structures.

Example 11

A topically-applied formulation of Peptide 14 is made including niacin,vitamin E, at least one preservative, at least one emulsifier andbetween 50-150 μM Peptide 14. The topical formulation was applied tohuman skin, resulting in the reduction of the appearance of wrinkles.

Example 12

A topically-applied formulation of Peptide 14 is made including niacin,vitamin E, at least one preservative, at least one emulsifier andbetween 75-100 μM Peptide 14. The topical formulation was applied tohuman skin, resulting in the reduction of the appearance of wrinkles.

Example 13

An exemplary topically-applied formulation is shown below in Table 4.The topical formulation was applied to human skin, resulting in thereduction of at least one of appearance of wrinkles, appearance of skintone (evenness), appearance of pores, appearance of texture andsmoothness, firmness, elasticity, and overall appearance. Thepolypeptides in the formulation comprises at least one of thepolypeptides disclosed herein.

TABLE 4 Exemplary Formulation Percentage in Components formulation Water 40%-65% Skin hydrators   5%-45% Emollients   6%-20% Vitamins 0.5%-5%Emulsifiers/stabilizers 0.5%-9% Polypeptides 0.001%-4% 

Example 14

An exemplary topically-applied formulation comprising Peptide 14 isshown below in Table 5. The topical formulation was applied to humanskin, resulting in the reduction of at least one of appearance ofwrinkles, appearance of skin tone (evenness), appearance of pores,appearance of texture and smoothness, firmness, elasticity, and overallappearance. The polypeptides in the formulation comprises Peptide 14.

TABLE 5 Exemplary Formulation Percentage in Components formulation Water 40%-65% Skin hydrators   5%-45% Emollients   6%-20% Vitamins 0.5%-5%Emulsifiers/stabilizers 0.5%-9% Polypeptides 0.001%-4% 

Example 15

Three-dimensional in vitro skin models and ex vivo human skin sampleswere assessed for skin aging after treatment with Peptide 14.

In vitro 3D skin model: 3D skin models were prepared using a modifiedmethod based on the preparation described in Pennacchi, P. C. et al.Glycated Reconstructed Human Skin as a Platform to Study thePathogenesis of Skin Aging. Tissue Eng. Part A 21, 2417-2425, 2015.Briefly, type I collagen gels embedded with fibroblasts were seeded withnormal human epidermal keratinocytes (NHEKs) on top of the gel andcultured for 24 hours in order for NHEKs to reach a monolayer. Then, thegels with NHEKs on the top were raised to an air-liquid interface andcultured for additional 10 days to allow for epidermal cornification.The gels were treated with 12.5 μM of Peptide 14 by addition of Peptide14 to the culture medium.

Ex-vivo human skin model: Skin samples from healthy human donors wereobtained from ZenBio (Research Triangle, NC) and maintained in anair-liquid interface culture with Dulbecco's Modified Eagle Medium(DMEM) (Invitrogen, Carlsbad, Calif.), supplemented with 10% (v/v) FBS.The skin samples were treated either with a control vehicle or 12.5 μMPeptide 14 in the media, on day one and day three. After five days, thesamples were harvested and fixed in formalin for histology or used forDNA isolation.

DNA methylation analysis: The predicted biological age, also referred toas molecular DNA age, was determined from the level of DNA methylationin 3D skin model samples and ex vivo human skin biopsy samples. TotalDNA samples were obtained from the samples using the QIAamp DNA Mini Kit(Qiagen) following manufacturer instructions. DNA methylationassessment, as a marker of skin aging, was performed using the humanIllumina Infinium EPIC 850K chip. DNA samples included: i) four skinbiopsy samples (from same donor) which were considered as untreatedcontrols, ii) four skin biopsy samples (same donor) treated with 12 μMPeptide 14, iii) three 3D skins samples (each sample a pool of 3 skinsfrom 1 donor, total 3 donors) which were considered as untreatedcontrols, iv) three 3D skins samples (each sample a pool of 3 skins from1 donor, total 3 donors) treated with 12 μM Peptide 14. The raw imagedata was processed using the commands preprocessRaw( ) followed bypreprocessSWAN( ). Methylation signals (M-values) were then converted toratios using the ratioConvert( ) and next to beta values using getBeta() all functions implemented in the “minfi” R package. Beta values werenormalized using the betaqn( ) method, which quantile normalizes betas,implemented by the “watermelon” package. and normalized using the“preprocessQuantile” normalization method implemented in the “minfi” Rpackage. Normalized beta values were used for age estimation.

Statistical analysis: Data was tested for normal distribution byShapiro-Wilk test. In cases where more than 2 groups were compared,one-way ANOVA was performed, followed by Bonferroni's multiplecomparisons test. For cases where paired samples were compared, a pairedt-test was performed. p≤0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism (GraphPadsoftware) or R software.

Results: The in vitro 3D skin models and ex vivo human skin samplesshowed a decrease in skin aging after 5 days of treatment with Peptide14 as shown in FIGS. 11A, 11B and 11C. FIG. 11A shows hematoxylin andeosin (H&E) stained histological images of the 3D skin equivalents (toprow) and ex vivo skin biopsy samples (bottom row) cultured with noPeptide 14 (Control) and 12.5 μM Peptide 14 for 5 days. The 3D skinequivalents and ex vivo skin samples treated with Peptide 14 generallyshowed epidermal thicknesses that are similar to or thicker than theuntreated control samples. FIG. 11B shows the predicted age, alsoreferred to as molecular DNA age, of the 3D skin model samples treatedwith 12.5 μM Peptide 14 (treatment) was lower than the predicted age forsamples that were untreated control (ctrl). The mean of predicted agefor the untreated 3D skin model was about 80 whereas the mean of thepredicted age for the Peptide 14-treated 3D skin model was about 66(p=0.25 by t-test). FIG. 11C shows the predicted age of ex vivo skinbiopsy treated with 12.5 μM Peptide 14 was lower than the predicted ageof samples that were untreated control (ctrl). The mean of predicted agefor the ex vivo skin biopsy samples was about 71 whereas the mean of thepredicted age for the Peptide 14-treated ex vivo skin biopsy samples wasabout 68 (**p<0.01).

Example 16

In vertebrates, specialized cells called melanocytes usually producemelanin. MeWo cells are a human immortalized melanocyte cell line thatrepresent late differentiated melanocytes expressing retinoic acidreceptors and may provide an experimental model for in vitroinvestigation of melanogenesis. Additional description regarding the useof MeWo cells as an in vitro model melanogenesis is found in Schadendorfet al., 1994. Retinoic Acid Receptor-gamma-selective Retinoids ExertAntiproliferative Effects on Human-melanoma Cell-growth In-vitro.International Journal of Oncology. Doi:10.3892/Ijo.5.6.1325 andMalaspina et al., Depigmenting potential of lichen extracts evaluated byin vitro and in vivo tests. PeerJ 8:e9150https://doi.org/10.7717/peerj.0.9150.

To test whether Peptide 14 promotes depigmentation of melanocytes, MeWocells were cultured in DMEM supplemented with 10% v/v FBS and 1% v/v ofpenicillin/streptomycin solution (1,000 U·mL⁻¹) at 5% CO₂, 37° C. and95% humidity. For two weeks prior to treatment with one of theexperimental conditions (pre-treatment), the MeWo cells were culturedwith isobutyl methylxanthine (IBMX) to stimulate melanin synthesis.After the MeWo cells were seeded in 6-well plates at 1,000,000 cells perwell for 6 hours, the cells were incubated with one of the experimentalconditions for 7 days and analyzed for intracellular and supernatantmelanin content.

The experimental conditions included a positive control group, anegative control group, a Peptide 14 (IBMX) 7d group, and a retinoicacid (IBMX) 7d group. The negative control group comprised MeWo cellswhich were stimulated with 25 μM IBMX for 2 weeks in the pre-treatmentperiod and then were untreated for 7 days, receiving the vehicle only,after being plated for the experiment. The positive control groupcomprised MeWo cells that were incubated with 25 μM IBMX during theentire experiment, for 2 weeks in the pre-treatment period and for 7days in the treatment period. The Peptide 14 (IBMX) 7d group comprisedMeWo cells that were incubated with 25 μM IBMX 2 weeks in thepre-treatment period and then treated for 7 days with 25 μM IBMX and3.12 μM Peptide 14. The retinoic acid (IBMX) 7d group comprised MeWocells that were incubated with 25 μM IBMX 2 weeks in the pre-treatmentperiod and then treated for 7 days with 25 μM IBMX and 2 μM retinoicacid.

After treatment, the melanin content in the cell pellet and the cellculture supernatant was assessed by the absorbance of the samples at 492nm. The cell pellet was prepared by trypsinization, counting andincubated for 16 hours in 200 μL of 1M NaOH (Matsuda et al., 2004; Yooet al., 2007). The cell culture supernatant was obtained after 1.2×gcentrifugation for 10 minutes. Data are presented as relative melanincontent, absorbance of the samples at a specified wavelength (492 nm)normalized to the absorbance of the negative control samples also as thesame wavelength. Three independent experiments including three technicalreplicates were performed. The data were analyzed using ANOVA andBonferroni post-hoc test when a statistical significance was detected.The statistically significant p-values are noted by * for p<0.05; ** forp<0.01; *** for p<0.001; **** for p<0.0001 between the groups.

The Peptide 14 treatment promoted a significant decrease in the relativemelanin content as compared to the positive control, negative controland retinoic acid treatment groups in the cellular pellet, as shown inFIG. 12A, and in the cell culture supernatant, as shown in FIG. 12B. Inthe cell pellets as shown in FIG. 12A, the positive control had arelative melanin content of about 170% times of the negative controlgroup (**** p<0.0001) and the retinoic acid treatment group had arelative melanin content of about 125% of the negative control group(**p<0.01) while Peptide 14 treatment had a relative melanin content ofabout 90% of the negative control group (***p<0.001) or lower than thenegative control group. In the cell culture supernatant as shown in FIG.12B, the positive control and the retinoic acid treatment group hadrelative melanin contents that were very similar to the negative controlgroup while Peptide 14 treatment had a relative melanin content of about70% of the negative control group (*p<0.05) or lower than the negativecontrol group.

This shows that Peptide 14 may have applications to reduce variousmelanogenesis-related conditions, including but not limited to melasma,skin hyperpigmentation caused by cutaneous inflammation, hormonalchanges, aging, sun exposure, and chronic lesions. In addition, Peptide14 appeared to perform better than retinoic acid, currently consideredthe gold standard molecule for anti-aging skincare.

Example 17

The skin frequently may experience rebound hyperpigmentation, wherepigmentation is reestablished in the skin after the discontinuation ofthe lightening treatment(s). To test whether the depigmentation effectof Peptide 14 remained after the Peptide 14 treatment was discontinued,MeWo cells were cultured in DMEM supplemented with 10% v/v FBS and 1%v/v of penicillin/streptomycin solution (1,000 U·mL⁻¹) at 5% CO₂, 37° C.and 95% humidity. For two weeks prior to treatment with one of theexperimental conditions (pre-treatment), the MeWo cells were culturedwith isobutyl methylxanthine (IBMX) to stimulate melanin synthesis.After the MeWo cells were seeded in 6-well plates at 1,000,000 cells perwell for 6 hours, the cells were incubated with one of the experimentalconditions for 14 days and analyzed for intracellular and supernatantmelanin content.

The experimental conditions included a positive control group, anegative control group, a Peptide 14 (IBMX) 14 days group, a retinoicacid (IBMX) 14 days group, a Peptide 14 (IBMX) interrupted on 7^(th) daygroup, and a retinoic acid (IBMX) interrupted on 7^(th) day group. Thenegative control group comprised MeWo cells which were stimulated with25 μM IBMX for 2 weeks in the pre-treatment period and then wereuntreated for 14 days, receiving the vehicle only, after being platedfor the experiment. The positive control group comprised MeWo cells thatwere incubated with 25 μM IBMX during the entire experiment, for 2 weeksin the pre-treatment period and for 14 days in the treatment period.Some of the groups were treated with both IBMX 25 μM for 7 or 14 days,as well as 3.12 μM Peptide 14, or both IBMX 25 μM (for 7 or 14 days), aswell as retinoic acid 2 μM. The Peptide 14 (IBMX) 14 day group comprisedMeWo cells that were incubated with 25 μM IBMX 2 weeks in thepre-treatment period and then treated for 14 days with 25 μM IBMX and3.12 μM Peptide 14. The retinoic acid (IBMX) 14 day group comprised MeWocells that were incubated with 25 μM IBMX 2 weeks in the pre-treatmentperiod and then treated for 14 days with 25 μM IBMX and 2 μM retinoicacid. The Peptide 14 (IBMX) 7 day group comprised MeWo cells that wereincubated with 25 μM IBMX 2 weeks in the pre-treatment period, thentreated for only 7 days with 25 μM IBMX and 3.12 μM Peptide 14, andcultured for additional 7 days with 25 μM IBMX only. The retinoic acid(IBMX) 7 day group comprised MeWo cells that were incubated with 25 μMIBMX 2 weeks in the pre-treatment period, then treated for only 7 dayswith 25 μM IBMX and 2 μM retinoic acid and cultured for additional 7days with 25 μM IBMX only.

After treatment, the melanin content in the cell pellet and the cellculture supernatant was assessed by the absorbance of the samples at 492nm. The cell pellet was prepared by trypsinization, counting andincubated for 16 hours in 200 μL of 1M NaOH (Matsuda et al., 2004; Yooet al., 2007). The cell culture supernatant was obtained after 1.2×gcentrifugation for 10 minutes. Data are presented as relative melanincontent, absorbance of the samples at a specified wavelength (492 nm)normalized to the normalized to the number of cells in the sampleabsorbance of the negative control samples also as the same wavelength.Since cell proliferation is significant during the 14 day period, inthis experiment only, the melanin content data was normalized to thenumber of cells in the sample within each group. Three independentexperiments including three technical replicates were performed. Thedata were analyzed using ANOVA and Bonferroni post-hoc test when astatistical significance was detected. The statistically significantp-values are noted by * for p<0.05; ** for p<0.01; *** for p<0.001; ****for p<0.0001 between the groups.

In addition, the mRNA level of key genes involved in melanogenesis,including tyrosinase, melanocyte inducing transcription factor (MITF)and dopachrome tautomerase (DCT) was analyzed.

The Peptide 14 treatment promoted a significant decrease in the relativemelanin content as compared to the positive control and retinoic acidtreatment groups in the cellular pellet, as shown in FIG. 13A, and inthe cell culture supernatant, as shown in FIG. 13B. In the cell pelletsas shown in FIG. 13A, the positive control had a relative melanincontent of about 130%, and the retinoic acid 14 day treatment group hada relative melanin content of about 130% while Peptide 14 14 daytreatment group had a relative melanin content of about 90%, or lowerthan the negative control group. The Peptide 14 7 day treatment grouphad a relative melanin content of about 40%, the lowest relative melanincontent of the experimental groups. The retinoic acid 7 day treatmentgroup had a relative melanin content of about 90%. In the cell culturesupernatant as shown in FIG. 13B, the positive control had a relativemelanin content of about 110%, while the retinoic acid 14 day treatmentgroup and the Peptide 14 14 day treatment group had relative melanincontents of about 50%. The retinoic acid 7 day treatment group and thePeptide 14 7 day treatment group had relative melanin contents of about100%.

The mRNA levels of melanogenesis-related genes are shown in FIGS. 14A,14B, and 14C. Peptide 14 treatment appears to result in significantdecreases in the expression of tyrosinase, MITF, and DCT genes, that arelower than or at least similar to the mRNA levels for retinoic acidtreated groups. As shown in FIG. 14A, the relative expression level oftyrosinase was lowest for Peptide 14 14 day treatment group as comparedto the other experimental groups. As shown in FIG. 14B, the relativeexpression level of MITF was the lowest for Peptide 14 14 day treatmentgroup and the retinoic acid 14 day treatment group. As shown in FIG.14C, the relative expression level of DCT was lowest for Peptide 14 14day treatment group and Peptide 14 7 day treatment group as compared tothe other experimental groups.

This shows that Peptide 14 may reduce melanogenesis and continue toresult in lower melanogenesis after discontinuing Peptide 14 treatment.In addition, Peptide 14 appeared to perform better than retinoic acid,currently considered the gold standard molecule for anti-aging skincare.

Example 18

To test the effect of Peptide 13 and Peptide 14 treatment on human skinmodels and the skin morphology, human primary fibroblasts andkeratinocytes isolated from healthy elder donors (71, 84, and 90 yearsold) were used to build human skin equivalents. The skin equivalentswere treated with 0.01% w/v (or 1 μM) of Peptide 13 or Peptide 14 for 5days. The negative controls were treated with formulation only. Afterthe 5-day culture, the skin equivalents were analyzed for epidermalthickness, which was quantified according to total epidermal area.Quality assessment based several parameters was performed by blindanalysts. The observed parameters include general organization of celllayers, as well as the thickness of the horny layer, among other aspectsand were shown to decrease with aging and senescence level. Theassessment had a maximum score of 28, where higher score correlated withdecrease in age and senescence. A minimal score of 19 was required forbatch use. This score was validated internally and shown to decreasewith age/senescence of the skin equivalents or cultured cells. Threeindependent experiments were performed with three technical replicates.Data were analyzed using a t-test. Statistical significance wasdetermined as p-values equal or lower than 0.05. The statisticallysignificant p-values are noted by * for p<0.05; ** for p<0.01; *** forp<0.001; **** for p<0.0001 between the groups.

FIG. 15A shows the H&E-stained histological images of the in vitro humanskin models treated with a vehicle only (control), Peptide 13, orPeptide 14. The human skin models treated with Peptide 13 or Peptide 14generally showed similar or increased thickness of stratum corneum layer(as indicated by dark gray layer on top of the images or stained brightpink by H&E) and epidermal layer (as shown by medium gray layer in themiddle of the images or stained purple by H&E) to the untreated controlsamples. FIG. 15B shows the mean of histology scores of human skinmodels treated with a vehicle only (control), Peptide 14, or Peptide 13,which were 21.00, 23.83, and 23.44, respectively. The histology scoreswere higher for samples treated with Peptide 13 or Peptide 14 than forthe negative control. Treatment of skin samples with Peptide 13 orPeptide 14 appeared to improve skin morphology as assessed by epidermalthickness and barrier.

Example 19

To study the level of penetration by Peptide 14 through the depth of theskin, a diffusion study was performed using Franz cells and on ex vivoskin culture sample. Fresh human skin from the abdomen of a female donor(79 years old) was cut in small pieces ˜2.5 cm×2.5 cm.

In the Franz cell study, the skin was treated with 10 μL of a formulatedcream comprising 0.01% Peptide 14 and placed in a Franz cell with acontact area of 5 mm diameter (0.2 cm²). The receptor chamber had 2 mLof PBS, pH 7.4. The Franz cells were kept for 24 hours at 32° C. underagitation.

In the ex vivo skin culture study, the skin was treated with 2 μL of aformulated cream comprising 0.01% of Peptide 14 (for a total of 200 ngof Peptide 14). The skin sample was then placed in an air-liquidinterface with DMEM media in the bottom and were kept for 24 hours at37° C.

After 24 hours, the excess formulation on the skin was removed with atissue paper, and the skin samples were washed 4 times in PBS. All thesurrounding skin was also removed. The skin was then incubated at 60° C.for 1-2 minutes to separate epidermis from dermis. The PBS in thereceptor chamber (2 mL), the epidermis layer, and the dermis layer werecollected and frozen at −80° C. until further analysis. The dermis layerwas analyzed mass spectrometry for the Peptide 14 to determine amount ofPeptide 14 that penetrated into the dermis.

In the Franz cell study, about 1.37% to 2.60% of the applied Peptide 14was found in the dermis layer. In the ex vivo skin culture study, about1.94% to 1.96% of the applied Peptide 14 was found in the dermis layer.Very little to no Peptide 14 penetrated into the dermis when Peptide 14was applied topically to the surface of the skin. This demonstrates thatPeptide 14 achieves very low to minimal dermal penetration with topicalapplication.

Example 20

A clinical study was performed on human subjects to assess the effect oftopical application of Peptide 14 on the face. This study was approvedby an IRB board. 22 human subjects participated in the clinical studyand were asked to use two products, one in each side of the face. On theright side of the face, the subjects used the negative controlcomprising the formulation only. On the left side of the face, thesubjects used the treatment formulation comprising 0.01% Peptide 14. Thesubjects underwent assessments before starting to use the product(baseline) and after 6 weeks and 12 weeks of daily topical applicationof the formulations to the respective sides of the face. Variousmeasures of skin moisture content, trans-epidermal water loss (TEWL),dermal thickness and echogenicity, intracutaneous analysis, skinviscoelastic properties, and skin surface profile were taken as detailedbelow to assess the effect of topical Peptide 14 application on facialskin on the appearance of lines/wrinkles, appearance of skin tone(evenness), appearance of pores, appearance of texture/smoothness,firmness (visual), elasticity (tactile), and overall appearance.

Clinical expert grading. Grading for Efficacy: Visual and tactileassessments were performed using 10 cm Visual Analog Scales (VAS) onbilateral face at baseline, week 6 and week 12. The following parameterswere evaluated: fine lines/wrinkle, skin tone (color evenness),texture/smoothness (visual), firmness (visual), elasticity (tactile),skin pores, radiance/luminosity, and overall appearance using theinstruments and methods detailed below.

Corneometer: Corneometer CM 825 (Courage+Khazaka, Germany) was used toevaluate skin moisture content/hydration by measuring skin capacitance.Measurements were taken in triplicate and averaged on bilateral face atbaseline, week 6 and week 12. A test site map was used to ensure thesame location is measured at each visit.

Vapo Meter: The VapoMeter (Delfin Technologies Ltd., Finland) measuresthe trans-epidermal water loss (TEWL) of the skin with a closedcylindrical chamber which has sensors to measure relative humidity andtemperature. Changes in TEWL rates provide a measure of barrierdisruption or integrity, thereby providing an indication of the effectof Peptide 14 on skin integrity. All subjects had VapoMeter measurementstaken in duplicate and averaged on the bilateral face at baseline, week6 and week 12. Assessment location was recorded on a body map for eachsubject.

Ultrasound—DermaScan: The DermaScan C USB (Cortex Technology ApS,Hadsund, Denmark) is a compact high-resolution ultrasound scanner. Allsubjects had ultrasound assessments taken on the bilateral face atbaseline and week 12. The location of assessments was the same at eachvisit and was recorded on a face map. Upon acquisition of the ultrasoundscans, they were analyzed for dermal thickness (density) andechogenicity.

SIAScope: The COSMETRICS™ SIAScope (Astron Clinical, Toft, UK) is anon-invasive optical skin imaging instrument using SpectrophotometricIntracutaneous Analysis (SIA) or chromophore mapping. Dermal collagenand hemoglobin were measured on the bilateral face at baseline, week 6and week 12.

Cutometer: The Cutometer MPA 580 (Courage+Khazaka, Germany) measures theviscoelastic properties of the skin (firmness and elasticity) byapplying suction to the skin surface, drawing the skin into the apertureof the probe and determining the penetration depth using an opticalmeasuring system. Measurements were taken on the bilateral face atbaseline, week 6 and week 12. The same location was measured at eachtime point and recorded using a face map. Measurements includedfirmness, elasticity and net elasticity.

VISTA-CR: Photo documentation was provided using the VISTA-CR imagingsystem (Canfield Scientific, Paramus, N.J., USA) which captureshigh-resolution images in multiple lighting modes. Photographs werecaptured in standard 1 and parallel polarized light of the center, rightand left view at baseline, week 6 and week 12.

ANTERA 3D®: The Antera 3D® (Miravex, Ireland) is an instrument combiningskin profilometry, multi-spectral analysis and colorimetry to providereconstruction of the skin surface in three dimensions and subsequentimage analysis. Images were captured on the crow's feet area on the leftand right in all subjects at baseline, week 6 and week 12. The samelocation was measured at each time point and recorded using a face map.Images were analyzed for texture, breadth and depth lines/wrinkles.

Expert Clinical Grading

Comparison of Week 6 and Week 12 to Baseline

Peptide 14 Formulation: Comparison of mean scores of the left side ofthe face treated with Peptide 14 at baseline to the subsequent timepoints revealed statistically significant improvements in clinicalgrading at week 6 which continued to week 12 for appearance oflines/wrinkles, skin tone (evenness), appearance of pores,texture/smoothness, firmness (visual), elasticity (tactile), and overallappearance. Additionally, the appearance of radiance/luminosity wasstatistically significantly improved at week 12 compared to the baselinefor left side of the face treated with Peptide 14.

Control Formulation: Comparison of mean scores of the right side of theface treated with the negative control formulation at baseline to thesubsequent time points revealed statistically significant improvementsin clinical grading at week 6 which continued to Week 12 for appearanceof lines/wrinkles, skin tone (evenness), appearance of pores,texture/smoothness, firmness (visual), elasticity (tactile),radiance/luminosity, and overall appearance.

Comparison of Peptide 14 Treatment to Negative Control (Left vs Rightside of face)

Comparison of Peptide 14 Treatment to Negative Control with TreatmentTime

When compared to baseline (before treatment), the left side of the facetreated with Peptide 14 appeared to be better than the right side of theface treated with the negative control in some of the measurements. Theleft side of the face treated with Peptide 14 had higher level of skinhydration by Corneometer measurement, higher level of collagen level bySIAScope measurements at week 6, and better TEWL (transepithelial waterloss) improvement by VapoMeter measurement at week 6 than the right sideof the face treated with the negative control. No significantdifferences were seen between the left side of the face treated withPeptide 14 and the right side of the face treated with the negativecontrol formulation for Dermascan assessment for dermal thickness(density) and echogenicity and Visia assessment for overall skinappearance. The left side of the face treated with Peptide 14 has betterAntera measurement of texture (roughness) at week 12 than the right sideof the face treated with the negative control formulation

The results of 22 patients after 12 weeks using a topical cream with0.01% Peptide 14 were analyzed. FIG. 16 shows an example of left side ofthe face treated with Peptide 14 at baseline (left, Baseline) and after12 weeks of treatment (right, 12 weeks). The 12 weeks of treatment with0.01% Peptide 14 appears to have decreased the appearance of lines andwrinkles and improved smoothness, skin tone (evenness),texture/smoothness, and overall appearance as compared to the baselinetime point.

An analysis of blind expert opinion was performed to determine thepercentage of patients showing improvement and mean percentageimprovement (MPI) of all subjects, where MPI represents the mean percentimprovement compared to baseline (before treatment). The analysis found87% of patients assessed as having a reduction in appearance of skinwrinkles (MPI: 3.3%), 90% assessed as having improved skin elasticity(MPI: 4.75%), and 95.5% assessed as having an improvement in skinevenness (MPI: 4.4%), radiance (MPI: 5.06%), appearance of pores (MPI:4.58%), and firmness (MPI: 5.43%). All subjects were assessed aspresenting better skin texture/smoothness (MPI: 7.46%) and overallappearance (MPI: 6.17%).

The faces of the subjects were evaluated by various instruments todetermine the percentages of patients showing improvement and meanpercentage improvement (MPI) of all subjects, where MPI represents themean percent improvement compared to baseline (before treatment). Theinstrumental evaluation found that 81% of the subjects had better skinbarrier according to Vapometer evaluation (MPI: 14.19%). 73% of thesubjects presented better skin roughness according to Antera evaluation(MPI: 5.05%). 73% of the subjects presented better skin radianceaccording to VISIA evaluation (MPI: 16.63%).

The perceptions of the subjects were also assessed. In the analysis ofsubjects' perception, greater than 80% of the subjects considered thatthe formulation comprising Peptide 14 promoted better skin appearance,better skin texture, better skin firmness, and better hydration. 78% ofthe subjects noticed an improvement in skin radiance.

Example 21

The topical formulation comprising at least one of the polypeptidesdisclosed herein is used on human skin. The formulation is directed tobe applied as a smooth layer onto clean, dry skin on face and/or neck inthe morning and the evening. The formulation is a daily essentialtopical supplement scientifically formulated to improve skin resilienceand strengthen epidermal barrier for long-lasting health of the skin.

Example 22

A user applies the topical formulation described herein comprising atleast one of the polypeptides to the face and/or neck. The user alsoapplies another topical formulation comprising a UV blocker afterapplying the topical formulation.

Example 23

A user applies the topical formulation described herein comprising atleast one of the polypeptides and a UV blocker to the face or the neck.The topical formulation may be applied to the skin on the body of theuser.

Example 24

3D skin equivalent sections of samples built with cells from elderdonors (71, 84 or 90 years old) were treated with 1 μM Peptide 14 orPeptide 13, or 20 μM Retinoic Acid (RA) and assessed for the effect ofthe various treatments of various markers of senescence, aging, andhealth.

FIG. 17 shows the relative mRNA expression levels of p16, BLIMPL ZYG11B,IL-8, Ki-67, ZIC1, MMP1, HAS2 of the epidermal (Epi) and dermal (Der)layers of 3D skin equivalents treated with a control, Peptide 14,Peptide 13, or Retinoic Acid. Data are presented as 2-ddCt normalized toGAPDH and untreated control. *p<0.05. Peptide 13 and Peptide 14-treatedsamples generally had similar relative mRNA expression levels for p16,BLIMP1, ZYG11B, IL-8, and Ki-67 in the epidermal layer, and for p16,MMP1, HAS2, IL-8, and Ki-67 in the dermal layer. Peptide 13 and Peptide14-treated samples generally had lower relative mRNA expression levelsthan RA-treated samples for p16, BLIMPL ZYG11B, IL-8, in the epidermallayer and for p16, MMP1, IL-8, and Ki-67 in the dermal layer. Peptide 13and Peptide 14-treated samples generally had higher relative mRNAexpression levels than RA-treated samples for Ki-67 in the epidermallayer and for HAS2 for the dermal layer. Peptide 13 and Peptide14-treated samples had similar relative mRNA expression levels asRA-treated samples for ZIC1 and Ki-67 for dermal layer.

Example 25

Senotherapeutic strategies can be linked to healthspan and lifespanextension in vivo. In order to assess whether Peptide 14 promotesextended healthspan and lifespan, Caenorhabditis elegans worms wereused. Peptide 14 was added in the worm media (M9 buffer media) atdifferent concentrations of 1 μM or 2 μM. Negative control wormsreceived vehicle only. Two healthspan parameters were assessed i)pharyngeal pumping and ii) worm movement. Lifespan was also determined.For pharyngeal pumping analysis, 15 worms had their pharynx movement(pumping) observed daily and counted for 20 seconds. This experiment wasrepeated 3 times, by 2 different blind observers, employing differentpopulations of animals at different days. Statistical difference wasdetected by analyzing each individual time point by One-Way ANOVA andDunnet's post hoc test. All groups were compared with the H₂O group. Forworm movement analysis, the basic movement of C. elegans, also calledthrashing, was measured daily for 15 worms. Observation duration was 30seconds. The experiment was repeated 3 times, by 2 different blindobservers, employing different populations of worms at different days.Statistical difference was detected by analyzing each individual timepoint by One-Way ANOVA and Dunnet's post hoc, and all groups werecompared with the H₂O group. For lifespan analysis, 15 worms had theirlifespan observed daily until the last worm died. This experiment wasrepeated 3 times, by 2 different blind observers, employing differentpopulations of worms at different days. The average lifespan wasmeasured. Statistical difference was detected by analyzing eachindividual time point by One-Way ANOVA and Dunnet's post hoc, and allgroups were compared with the H₂O group (FIG. 18).

Treatment with either 1 μM or 2 μM of Peptide 14 improved worm thrashing(FIG. 18, panel A), pumping (FIG. 18, panel B), and lifespan (FIG. 18,panel C). While thrashing was significantly improved in day 1 (*p<0.05),pumping was significantly decreased at this day (*p<0.5). Takentogether, the data suggest safety and efficacy of the tested peptideswith regards to promoting healthspan and lifespan. For instance, on day8, both thrashing (*p<0.05) and pumping (*p<0.05) were increased in thegroups treated with Peptide 14 compared to control. Pumping decreaseswith aging in C. elegans, mainly as a result of muscular integrity loss.Even though not directly measured, defects in the macroscopic anatomy ofC. elegans' pharynx (bent or swollen pharynx, both common features ofthis nematode aging) were also reduced in the 1 μM Peptide 14 group whencompared to the H₂O control group. No decrease in worm movement wasdetected in either time point or sample, suggesting that the peptide wasnot toxic to C. elegans at the concentrations tested. When used at 1 μMand 2 μM, Peptide 14 promoted a statistically significant increase inworm average lifespan (1 μM Peptide 14 **p<0.01; 2 μM peptide14*p<0.05).

Example 26

Skin cells from Progeria patients were used as a model of aging becauseof the high levels of cellular senescence. Primary fibroblasts fromProgeria patients were cultured in DMEM (Dulbecco's Modified Eagle'sMedium) supplemented with 10% v.v. fetal bovine serum (FBS), and 1% v.v.of penicillin/streptomycin solution (1,000 U·mL−1). Cells cultured at 5%CO₂, 37° C. and 95% humidity atmosphere. After expansion, the cells wereseeded in 96-well plates (4,000 cells per well) and, 6 hours afterplating, were incubated with 500 nM, 5 or 50 μM of polypeptide sequenceLKGIL (SEQ ID NO: 6) or WLKGI (SEQ ID NO: 7). Negative control compriseduntreated cells, which received vehicle only. After incubation, relativecellular senescence, which was assessed by the activity of senescenceassociated β-galactosidase staining relative to untreated control and bythe quantification of ATRX foci/nuclei, was analyzed. Three independentexperiments (biological replicates) including three technical replicateswere performed. Data was analyzed using ANOVA and a Bonferroni post-hoctest. Statistical significance was determined as p values equal or lowerthan 0.05, where *p<0.05 and ** p<0.01.

FIG. 19 shows the effect of polypeptide sequences LKGIL (SEQ ID NO: 6)(A, B, C), and WLKGI (SEQ ID NO: 7) (D, E, F) to decrease cellularsenescence without promoting cell death. In panels A and D, the y-axisindicates the relative senescence level normalized to untreated control.In panels B and D, y-axis indicates the relative cell number normalizedto untreated control. In panels C and F, y-axis indicates the averageATRX foci accumulation per cell. Treatment of cells with LKGIL (SEQ IDNO: 6) significantly decreased senescence associated β-galactosidasestaining and the average number of ATRX foci/nuclei, when used at 5 μMand 50 compared to untreated cells (p<0.01 for β-galactosidase stainingand p<0.05 for ATRX foci/nuclei). Treatment of cells with WLKGI (SEQ IDNO: 7) decreased senescence associated β-galactosidase staining levelswhen used at 5 μM and 50 μM as compared to untreated cells (p<0.05 for 5μM and p<0.01 for 50 No cellular toxicity was observed in the testedconcentrations.

Example 27

Additional polypeptides were tested for suitability as an anti-senescentagent. Cells were incubated with one of the polypeptides. Negativecontrol comprised untreated cells, which received vehicle only. Afterincubation, relative cellular senescence (assessed by the activity ofsenescence associated β-galactosidase staining relative to negativecontrol) and relative cell proliferation relative to negative controlwere analyzed. A number of polypeptides showed relative cellularsenescence lower than 1, having decreased cellular senescence than theuntreated negative control, and maintained cell proliferation at orabove that of the untreated negative control. Examples of suchpolypeptides shown in Table 6.

Further, a dose-dependent effects of polypeptides of Table 6 on cellsenescence were studied. The cells were incubated various doses of thepolypeptides ranging from 1.26 μM to 50 μM (1.26 μM, 3.12 μM, 6.25 μM,12.5 μM, 25 μM, 50 μM). There was noticeable decrease in cell senescenceeven at lower doses for the polypeptides.

TABLE 6 Polypeptides SEQ ID NO. Polypeptide sequence 9 STAKAWLKGI 10ETAKAWEKGI 11 ETAKAWHKGI 12 ETAKAWLKSI 13 ETAKAWLKGE 14 ETAKAWLKGI 15WLKGILRGAA 16 KTAKAWLKGI 17 NTAKAWLKGI 18 PTAKAWLKGI 19 QTAKAWLKGI 20EQAKAWLKGI 21 WLKGICRGAA 22 WLKGILPGAA 23 WLKGILQGAA 24 WLKGILSGAA 25WLKGILVGAA 26 WLKGILRAAA 27 WLKGILRGHA 28 WLKGILRGIA

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Exemplary Embodiments

Among the exemplary embodiments are:

Embodiment 1 comprises compositions for treatment of skin comprising anisolated, synthetic, or recombinant polypeptide comprising an amino acidsequence of LKGI (SEQ ID NO:5) or an analog thereof, wherein in thepolypeptide comprises no more than 100 amino acids.

Embodiment 2. The compositions of embodiment 1, wherein the isolated,synthetic, or recombinant polypeptide comprises an amino acid sequenceof WLKGI (SEQ ID NO:7) or an analog thereof. Embodiment 3. Thecompositions of embodiment 1 or embodiment 2, wherein the isolated,synthetic, or recombinant polypeptide comprises an amino acid sequenceof LKGIL (SEQ ID NO:6) or an analog thereof. Embodiment 4. Thecompositions of any one of embodiments 1-3, wherein the isolated,synthetic, or recombinant polypeptide comprises at least 4 amino acids,10 amino acids, 15 amino acids, or 20 amino acids. Embodiment 5. Thecompositions of any one of embodiments 1-4, further comprising atherapeutic, nutraceutical, or cosmetic excipient. Embodiment 6. Thecompositions of embodiment 5, wherein the excipient is configured fortopical application. Embodiment 7. The compositions of any one ofembodiments 1-6, wherein the compositions are configured for applicationto human skin. Embodiment 8. The compositions of any one of embodiments1-7, wherein the compositions is a cream, a transdermal patch, a topicalpatch, an ointment, an oil, a gel, a liquid, a powder, a lotion, aserum, an emulsion, a moisturizer, a foam, a face mask, a mousse, anaerosol, a spray, a cleanser, a toner, or a shampoo. Embodiment 9. Thecompositions of any one of embodiments 1-5, wherein the compositions areconfigured as an edible supplement. Embodiment 10. The compositions ofembodiment 9, wherein the compositions is configured as a beverage.Embodiment 11. The compositions of any one of embodiments 1-9, whereinthe compositions comprise at least one skin hydrating agent selectedfrom a group consisting of glycerin, squalene, sorbitol, hyaluronicacid, hyaluronic acid derivatives, sodium hyaluronate, sodiumhyaluronate crosspolymer, niacinamide, glycoproteins, pyrrolidonecarboxylic acid (PCA), lysine HCl, allantoin and algae extract.Embodiment 12. The compositions of any one of embodiments 1-9, whereinthe compositions comprise at least one emollient selected from a groupconsisting of plant oils, mineral oil, shea butter, cocoa butter,petrolatum, fatty acids, triglycerides, benzoates, myristates,palmitates, stearates, glycolipids, phospholipids, squalene, glycerin,ceramide, and algae extract. Embodiment 13. The compositions ofembodiment 12, wherein the plant oil is selected from a group consistingof rose hip oil, andiroba oil, grape seed oil, avocado oil, plum seedoil, pracaxi oil, Calycophyllum spruceanum oil, almond oil, and arganoil. Embodiment 14. The compositions of any one of embodiments 1-13,wherein the compositions comprises at least one vitamin selected from agroup consisting of vitamin A, vitamin D, vitamin E, vitamin F, vitaminK, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin),vitamin B5 (pantothenic acid), vitamin B7 (biotin), vitamin B6, vitaminB12 (cyanocobalamin), vitamin B9, folic acid, niacinamide, orderivatives thereof. Embodiment 15. The compositions of any one ofembodiments 1-14, wherein the compositions comprise an effective amountof the isolated, synthetic, or recombinant polypeptide of about 500 nMto about 500 μM. Embodiment 16. The compositions of any one ofembodiments 1-14, wherein the compositions comprise an effective amountof the isolated, synthetic, or recombinant polypeptide of about 0.001%to about 5% (w/w). Embodiment 17. The compositions of any one ofembodiments 1-16, wherein the compositions are formulated to achieve lowdermal penetration of no more than 10% of the isolated, synthetic, orrecombinant polypeptide into the dermis. Embodiment 18. The compositionsof any one of embodiments 1-17, wherein the compositions improve atleast one of skin moisture content, trans-epidermal water loss (TEWL),dermal thickness and echogenicity, intracutaneous analysis, skinviscoelastic properties, or skin surface profile of skin of a user afterusing compositions on the skin as compared to before using thecompositions. Embodiment 19. The compositions of any one of embodiments1-17, wherein the compositions reduces appearance of lines/wrinkles,appearance of skin tone (evenness), appearance of pores, appearance oftexture/smoothness, firmness (visual), elasticity (tactile), epidermalbarrier, skin roughness, skin hyperpigmentation, or overall appearanceof skin of a user after using compositions on the skin as compared tobefore using the compositions. Embodiment 20. The compositions of anyone of embodiments 1-19, wherein the treatment of skin with thecomposition reduces or treats an effect of aging on the skin. Embodiment21. The compositions of any one of embodiments 1-20, wherein treatmentof skin with the composition reduces an effect of cellular senescence onthe skin. Embodiment 22. The compositions of any one of embodiments20-21, wherein the effect of aging is assessed by at least one ofsenescence associated β-galactosidase activity level, a ratio of ATRXfoci/cell, p16 expression, IL-8 expression, Ki-67 expression, hyaluronicsynthase 2 expression, matrix metalloprotease 1 (MMP1) expression,sirtuin 6 (SIRT6) expression, BLM expression, or exonuclease 1 (EXO1)expression.

Embodiment 23 comprises an isolated, synthetic, or recombinantpolypeptide comprising an amino acid sequence of X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀or an analog thereof, wherein: (a) the amino acid sequence has at least70%, 80%, 85%, 90%, or 95% identity to a first sequence SEQ ID NO:1wherein X₁ is E, X₂ is T, X₄ is K, X₆ is W, X₇ is L, X₉ is G, and X₁₀ isI; and (i) X₃ is not S (SEQ ID NO: 8); or (ii) if X₅ is any amino acidthen X₈ is not G (SEQ ID NO: 29); or (iii) if X₈ is any amino acid thenX₅ is not N (SEQ ID NO: 30); or (iv) any one of (i), (ii), or (iii)optionally with 1, 2, 3, or 4 conservative amino acid substitutions; or(b) the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95%identity to a second sequence SEQ ID NO:2 wherein X₁ is A, X₂ is T, X₃is A, X₄ is K, X₅ is A, X₆ is W, X₇ is L, X₈ is K, X₉ is G, and X₁₀ isI, optionally with 1, 2, 3, or 4 conservative amino acid substitutions;or (c) the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95%identity to a third sequence SEQ ID NO:3 wherein X₁ is K, X₂ is L, X₅ isI, X₆ is L, X₈ is G, and X₁₀ is A; and (i) if X₉ is any amino acid thenX₃ is not N (SEQ ID NO: 31); or (ii) if X₃ is any amino acid then X₉ isnot S (SEQ ID NO: 32); or (iii) if X₄ is any amino acid then X₇ is not L(SEQ ID NO: 33); or (iv) if X₇ is any amino acid then X₄ is not S (SEQID NO: 34), and; or (v) any one of (i), (ii), (iii), or (iv) optionallywith 1, 2, 3, or 4 conservative amino acid substitutions; or (d) theamino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity toa fourth sequence SEQ ID NO:4 wherein X₁ is W, X₂ is L, X₃ is K, X₄ isG, X₅ is I, X₆ is L, X₇ is R, X₈ is E, X₉ is A, and X₁₀ is A, optionallywith 1, 2, 3, or 4 conservative amino acid substitutions. Embodiment 24.The polypeptide of embodiment 23, wherein the amino acid sequencecomprises LKGI (SEQ ID NO:5). Embodiment 25. The polypeptide ofembodiment 23 or embodiment 24, wherein the amino acid sequencecomprises WLKGI (SEQ ID NO:7). Embodiment 26. The polypeptide of any oneof embodiments 23-25, wherein the amino acid sequence comprises LKGIL(SEQ ID NO:6). Embodiment 27. The polypeptide of embodiment 23, whereinthe amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identityto a sequence of SEQ ID NO:1. Embodiment 28. The polypeptide ofembodiment 23, wherein the amino acid sequence is SEQ ID NO:1.Embodiment 29. The polypeptide of embodiment 23, wherein the amino acidsequence is SEQ ID NO:2. Embodiment 30. The polypeptide of embodiment23, wherein the amino acid sequence has at least 70%, 80%, 85%, 90%, or95% identity to a sequence of SEQ ID NO:3. Embodiment 31. Thepolypeptide of embodiment 23, wherein the amino acid sequence has atleast 80%, 85%, 90%, or 95% identity to a sequence of SEQ ID NO:3.Embodiment 32. The polypeptide of embodiment 23, wherein the amino acidsequence is SEQ ID NO:3. Embodiment 33. The polypeptide of embodiment23, wherein the amino acid sequence is SEQ ID NO:4. Embodiment 34. Thepolypeptide of any one of embodiments 23-33, wherein the recombinantpolypeptide comprises at least 10 amino acids, 15 amino acids, or 20amino acids and no more than 100 amino acids. Embodiment 35 comprisescompositions for treatment of skin comprising at least one isolated,synthetic, or recombinant polypeptide of any one of embodiments 23-34and a therapeutic, nutraceutical, or cosmetic excipient. Embodiment 36.The compositions of embodiment 35, wherein the excipient is configuredfor topical application. Embodiment 37. The compositions of embodiment35 or embodiment 36, wherein the compositions is formulated forapplication to human skin. Embodiment 38. The compositions of any one ofembodiments 35-37, wherein the compositions is a cream, an ointment, agel, a liquid, an oil, a powder, a lotion, a serum, an emulsion, amoisturizer, a foam, a face mask, a mousse, an aerosol, a spray, acleanser, a toner, a topical patch, or a shampoo. Embodiment 39. Thecompositions of embodiment 35, wherein the compositions is configured asan edible supplement. Embodiment 40. The compositions of embodiment 39,wherein the compositions is configured as a beverage.

Embodiment 41. The compositions of any one of embodiments 35-40, whereinthe compositions comprise at least one skin hydrating agent selectedfrom a group consisting of glycerin, squalene, sorbitol, hyaluronicacid, hyaluronic acid derivatives, sodium hyaluronate, sodiumhyaluronate crosspolymer, niacinamide, glycoproteins, pyrrolidonecarboxylic acid (PCA), lysine HCl, allantoin and algae extract.Embodiment 42. The compositions of any one of embodiments 35-40, whereinthe compositions comprise at least one emollient selected from a groupconsisting of plant oils, mineral oil, shea butter, cocoa butter,petrolatum, fatty acids, triglycerides, benzoates, myristates,palmitates, stearates, glycolipids, phospholipids, squalene, glycerin,ceramide, and algae extract. Embodiment 43. The compositions ofembodiment 42, wherein the plant oil is selected from a group consistingof rose hip oil, andiroba oil, grape seed oil, avocado oil, plum seedoil, pracaxi oil, Calycophyllum spruceanum oil, almond oil, and arganoil. Embodiment 44. The compositions of any one of embodiments 35-43,wherein the compositions comprises at least one vitamin selected from agroup consisting of vitamin A, vitamin D, vitamin E, vitamin F, vitaminK, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin),vitamin B5 (pantothenic acid), vitamin B7 (biotin), vitamin B6, vitaminB12 (cyanocobalamin), vitamin B9, folic acid, niacinamide, orderivatives thereof. Embodiment 45. The compositions of any one ofembodiments 35-44, wherein the compositions comprise an effective amountof the isolated, synthetic, or recombinant polypeptide of about 500 nMto about 500 μM. Embodiment 46. The compositions of any one ofembodiments 35-44, wherein the compositions comprise an effective amountof the isolated, synthetic, or recombinant polypeptide of about 0.001%to about 5% (w/w). Embodiment 47. The compositions of any one ofembodiments 35-44, wherein the compositions comprises an effectiveamount of the isolated, synthetic or recombinant polypeptide of about0.001% to about 1%. Embodiment 48. The compositions of any one ofembodiments 35-47, wherein the compositions improve at least one of skinmoisture content, trans-epidermal water loss (TEWL), dermal thicknessand echogenicity, intracutaneous analysis, skin viscoelastic properties,or skin surface profile of skin of a user after using compositions onthe skin as compared to before using the compositions. Embodiment 49.The compositions of any one of embodiments 35-47, wherein thecompositions reduces appearance of lines/wrinkles, appearance of skintone (evenness), appearance of pores, appearance of texture/smoothness,firmness (visual), elasticity (tactile), epidermal barrier, skinroughness, skin hyperpigmentation, or overall appearance of skin of auser after using compositions on the skin as compared to before usingthe compositions. Embodiment 50. The compositions of any one ofembodiments 35-49, wherein the treatment of skin with the compositionreduces or treats an effect of aging on the skin. Embodiment 51. Thecompositions of any one of embodiments 35-50, wherein the treatment ofskin with the composition reduces an effect of cellular senescence onthe skin. Embodiment 52. The compositions of any one of embodiments50-51, wherein the effect of aging is assessed by at least one ofsenescence associated B-galactosidase activity level, a ratio of ATRXfoci/cell, p16 expression, IL-8 expression, Ki-67 expression, hyaluronicsynthase 2 expression, matrix metalloprotease 1 (MMP1) expression,sirtuin 6 (SIRT6) expression, BLM expression, or exonuclease 1 (EXO1)expression.

Embodiment 53 comprises methods of treating a condition in a subject inneed thereof, the method comprising administering to the subject acomposition comprising an amino acid sequence of SEQ ID NO:5. Embodiment54. The method of embodiment 53, wherein the formulation comprises anamino acid sequence of SEQ ID NO:6. Embodiment 55. The method ofembodiment 53 or embodiment 54, wherein the composition comprises anamino acid sequence of SEQ ID NO:7. Embodiment 56. The method of any oneof embodiments 53-55, wherein the administering comprises topicallyapplying the compositions to the subject. Embodiment 57. The method ofany one of embodiments 53-56, wherein the subject is a human or otheranimal. Embodiment 58. The method of any one of embodiments 53-57,wherein the method comprises administering an effective amount of thecompositions to the subject. Embodiment 59. The method of any one ofembodiments 53-58, wherein the condition is a disorder associated withaccumulation of senescent cells in the subject. Embodiment 60. Themethod of embodiment 53, wherein the disorder associated withaccumulation of senescent cells comprises aging skin. Embodiment 61. Themethod of any one of embodiments 53-60, wherein the compositionscomprises at least one skin hydrating agent selected from a groupconsisting of glycerin, squalene, sorbitol, hyaluronic acid, hyaluronicacid derivatives, sodium hyaluronate, sodium hyaluronate crosspolymer,niacinamide, glycoproteins, pyrrolidone carboxylic acid (PCA), lysineHCl, allantoin and algae extract. Embodiment 62. The method of any oneof embodiments 53-61, wherein the compositions comprises at least oneemollient selected from a group consisting of plant oils, mineral oil,shea butter, cocoa butter, petrolatum, fatty acids, triglycerides,benzoates, myristates, palmitates, stearates, glycolipids,phospholipids, squalene, glycerin, ceramide, and algae extract.Embodiment 63. The method of embodiment 62, wherein the plant oil isselected from a group consisting of rose hip oil, andiroba oil, grapeseed oil, avocado oil, plum seed oil, pracaxi oil, Calycophyllumspruceanum oil, almond oil, and argan oil. Embodiment 64. The method ofany one of embodiments 53-63, wherein the compositions comprises atleast one vitamin selected from a group consisting of vitamin A, vitaminD, vitamin E, vitamin F, vitamin K, vitamin B1 (thiamine), vitamin B2(riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid),vitamin B7 (biotin), vitamin B6, vitamin B12 (cyanocobalamin), vitaminB9, folic acid, niacinamide, or derivatives thereof. Embodiment 65. Themethod of any one of embodiments 53-64, wherein the compositionscomprises an effective amount of the isolated, synthetic, or recombinantpolypeptide of about 500 nM to about 500 μM. Embodiment 66. The methodof any one of embodiments 53-64, wherein the compositions comprises aneffective amount of the isolated, synthetic, or recombinant polypeptideof about 0.001% to about 5% (w/w). Embodiment 67. The method of any oneof embodiments 53-66, wherein the compositions is formulated to achievelow dermal penetration of no more than 10% of the isolated, synthetic,or recombinant polypeptide into the dermis. Embodiment 68. The method ofany one of embodiments 53-67, wherein the compositions improves at leastone of skin moisture content, trans-epidermal water loss (TEWL), dermalthickness and echogenicity, intracutaneous analysis, skin viscoelasticproperties, or skin surface profile of skin of a user after usingcompositions on the skin as compared to before using the compositions.Embodiment 69. The method of any one of embodiments 53-68, wherein thecompositions reduces appearance of lines/wrinkles, appearance of skintone (evenness), appearance of pores, appearance of texture/smoothness,firmness (visual), elasticity (tactile), epidermal barrier, skinroughness, skin hyperpigmentation, or overall appearance of skin of auser after using compositions on the skin as compared to before usingthe compositions. Embodiment 70. The method of any one of embodiments53-69, wherein treating a skin with the composition reduces or treats aneffect of aging on the skin. Embodiment 71. The method of any one ofembodiments 53-70, wherein treating the skin with the compositionreduces an effect of cellular senescence on the skin. Embodiment 72. Themethod of any one of embodiments 70-71, wherein the effect of aging isassessed by at least one of senescence associated B-galactosidaseactivity level, a ratio of ATRX foci/cell, p16 expression, IL-8expression, Ki-67 expression, hyaluronic synthase 2 expression, matrixmetalloprotease 1 (MMP1) expression, sirtuin 6 (SIRT6) expression, BLMexpression, or exonuclease 1 (EXO1) expression.

Embodiment 73. A method of reducing cellular senescence in a subject inneed thereof, the method comprising administering to the subject acomposition comprising a polypeptide comprising an amino acid sequenceof LKGI (SEQ ID NO:5) optionally with 1 conservative amino acidsubstitution, wherein in the polypeptide comprises no more than 100amino acids. Embodiment 74. The method of embodiment 73, wherein thepolypeptide comprises at least 4 amino acids, 10 amino acids, 15 aminoacids, or 20 amino acids. Embodiment 75. The method of embodiment 73,wherein the compositions comprises an amino acid sequence of WLKGI (SEQID NO:7) optionally with 1 conservative amino acid substitution.Embodiment 76. The method of embodiment 73, wherein the compositionscomprises an amino acid sequence of LKGIL (SEQ ID NO:6) optionally with1 conservative amino acid substitution. Embodiment 77. The method ofembodiment 75 or embodiment 76, wherein the polypeptide comprises atleast 5 amino acids, 10 amino acids, 15 amino acids, or 20 amino acidsand comprises no more than 100 amino acids. Embodiment 78. The method ofany one of embodiments 73-77, wherein the compositions further comprisesa therapeutic, nutraceutical, or cosmetic excipient. Embodiment 79. Amethod of reducing cellular senescence in a subject in need thereof, themethod comprising administering to the subject a therapeutic,nutraceutical, or cosmetic compositions comprising at least onepolypeptide of any one of embodiments 23-34. Embodiment 80. The methodof embodiment 79, wherein the compositions further comprises atherapeutic, nutraceutical, or cosmetic excipient. Embodiment 81. Themethod of embodiment 79 or embodiment 80, wherein the administeringcomprises applying the compositions to a portion of the skin of thesubject. Embodiment 82. The method of any one of embodiments 79-81,wherein the compositions extends a lifespan of a plurality of cells ofthe subject, induces SIRT6 expression in a plurality of cells of thesubject, increases cell renewal rates in a plurality of cells of thesubject, promotes apoptosis in a plurality of cells of the subject,promotes DNA repair in a plurality of cells of the subject, increasescollagen production in a plurality of cells of the subject, increaseshyaluronic synthase production in a plurality of cells of the subject,decreases ATRX nuclear foci accumulation in a plurality of cells of thesubject, decreases p16 expression in a plurality of cells of thesubject, decreases senescence associated beta-galactosidase productionin a plurality of cells of the subject, decreases IL8 expression in aplurality of cells of the subject, decreases MMP1 expression in aplurality of cells of the subject, increases BLM expression in aplurality of cells of the subject, and/or prevents UV-induced DNA damagein a plurality of cells of the subject.

Embodiment 83 comprises methods of treating a condition in a subject inneed thereof, the method comprising administering to the subject atherapeutic, nutraceutical, or cosmetic compositions comprising an aminoacid sequence of SEQ ID NO:5 optionally with 1 conservative amino acidsubstitution. Embodiment 84. The method of embodiment 83, wherein thetherapeutic, nutraceutical, or cosmetic compositions comprises an aminoacid sequence of SEQ ID NO:6 optionally with 1 conservative amino acidsubstitution. Embodiment 85. The method of embodiment 83 or embodiment84, wherein the therapeutic, nutraceutical, or cosmetic compositionscomprises an amino acid sequence of SEQ ID NO:7 optionally with 1conservative amino acid substitution.

1. A composition for treatment of skin comprising an isolated,synthetic, or recombinant polypeptide comprising an amino acid sequenceof LKGI (SEQ ID NO:5) or an analog thereof, wherein in the polypeptidecomprises no more than 100 amino acids.
 2. The composition of claim 1,wherein the isolated, synthetic, or recombinant polypeptide comprises anamino acid sequence of WLKGI (SEQ ID NO:7) or an analog thereof.
 3. Thecomposition of claim 1, wherein the isolated, synthetic, or recombinantpolypeptide comprises an amino acid sequence of LKGIL (SEQ ID NO:6) oran analog thereof.
 4. The composition of claim 1, wherein the isolated,synthetic, or recombinant polypeptide comprises at least 4 amino acids,10 amino acids, 15 amino acids, or 20 amino acids.
 5. The composition ofclaim 1, further comprising a therapeutic, nutraceutical, or cosmeticexcipient.
 6. The composition of claim 5, wherein the excipient isconfigured for topical application.
 7. (canceled)
 8. The composition ofclaim 6, wherein the composition is a cream, a transdermal patch, atopical patch, an ointment, an oil, a gel, a liquid, a powder, a lotion,a serum, an emulsion, a moisturizer, a foam, a face mask, a mousse, anaerosol, a spray, a cleanser, a toner, or a shampoo.
 9. (canceled) 10.(canceled)
 11. The composition of claim 1, wherein the compositioncomprises at least one skin hydrating agent selected from a groupconsisting of glycerin, squalene, sorbitol, hyaluronic acid, hyaluronicacid derivatives, sodium hyaluronate, sodium hyaluronate crosspolymer,niacinamide, glycoproteins, pyrrolidone carboxylic acid (PCA), lysineHCl, allantoin and algae extract.
 12. The composition of claim 1,wherein the composition comprises at least one emollient selected from agroup consisting of plant oils, mineral oil, shea butter, cocoa butter,petrolatum, fatty acids, triglycerides, benzoates, myristates,palmitates, stearates, glycolipids, phospholipids, squalene, glycerin,ceramide, and algae extract.
 13. (canceled)
 14. The composition of claim1, wherein the composition comprises at least one vitamin selected froma group consisting of vitamin A, vitamin D, vitamin E, vitamin F,vitamin K, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3(niacin), vitamin B5 (pantothenic acid), vitamin B7 (biotin), vitaminB6, vitamin B12 (cyanocobalamin), vitamin B9, folic acid, niacinamide,or derivatives thereof.
 15. The composition of claim 1, wherein thecomposition comprises an effective amount of the isolated, synthetic, orrecombinant polypeptide of about 500 nM to about 500 μM.
 16. Thecomposition of claim 1, wherein the composition comprises an effectiveamount of the isolated, synthetic, or recombinant polypeptide of about0.001% to about 5% (w/w).
 17. (canceled)
 18. The composition of claim 1,wherein the composition improves at least one of skin moisture content,trans-epidermal water loss (TEWL), dermal thickness and echogenicity,intracutaneous analysis, skin viscoelastic properties, or skin surfaceprofile of skin of a user after using composition on the skin ascompared to before using the composition.
 19. The composition of claim1, wherein the composition reduces appearance of lines/wrinkles,appearance of skin tone (evenness), appearance of pores, appearance oftexture/smoothness, firmness (visual), elasticity (tactile), epidermalbarrier, skin roughness, skin hyperpigmentation, or overall appearanceof skin of a user after using composition on the skin as compared tobefore using the composition.
 20. The composition of claim 1, whereinthe treatment of skin with the composition reduces or treats an effectof aging on the skin.
 21. The composition of claim 1, wherein treatmentof skin with the composition reduces an effect of cellular senescence onthe skin.
 22. The composition of claim 1, wherein the effect of aging isassessed by at least one of senescence associated β-galactosidaseactivity level, a ratio of ATRX foci/cell, p16 expression, IL-8expression, Ki-67 expression, hyaluronic synthase 2 expression, matrixmetalloprotease 1 (MMP1) expression, sirtuin 6 (SIRT6) expression, BLMexpression, or exonuclease 1 (EXO1) expression.
 23. An isolated,synthetic, or recombinant polypeptide comprising an amino acid sequenceof X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀ or an analog thereof, wherein: (a) the aminoacid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to afirst sequence SEQ ID NO:1 wherein X₁ is E, X₂ is T, X₄ is K, X₆ is W,X₇ is L, X₉ is G, and X₁₀ is I; and (i) X₃ is not S (SEQ ID NO: 8); or(ii) if X₅ is any amino acid then X₈ is not G (SEQ ID NO: 29); or (iii)if X₈ is any amino acid then X₅ is not N (SEQ ID NO: 30); or (iv) anyone of (i), (ii), or (iii) optionally with 1, 2, 3, or 4 conservativeamino acid substitutions; or (b) the amino acid sequence has at least70%, 80%, 85%, 90%, or 95% identity to a second sequence SEQ ID NO:2wherein X₁ is A, X₂ is T, X₃ is A, X₄ is K, X₅ is A, X₆ is W, X₇ is L,X₈ is K, X₉ is G, and X₁₀ is I, optionally with 1, 2, 3, or 4conservative amino acid substitutions; or (c) the amino acid sequencehas at least 70%, 80%, 85%, 90%, or 95% identity to a third sequence SEQID NO:3 wherein X₁ is K, X₂ is L, X₅ is I, X₆ is L, X₈ is G, and X₁₀ isA; and (i) if X₉ is any amino acid then X₃ is not N (SEQ ID NO: 31); or(ii) if X₃ is any amino acid then X₉ is not S (SEQ ID NO: 32); or (iii)if X₄ is any amino acid then X₇ is not L (SEQ ID NO: 33); or (iv) if X₇is any amino acid then X₄ is not S (SEQ ID NO: 34), and; or (v) any oneof (i), (ii), (iii), or (iv) optionally with 1, 2, 3, or 4 conservativeamino acid substitutions; or (d) the amino acid sequence has at least70%, 80%, 85%, 90%, or 95% identity to a fourth sequence SEQ ID NO:4wherein X₁ is W, X₂ is L, X₃ is K, X₄ is G, X₅ is I, X₆ is L, X₇ is R,X₈ is E, X₉ is A, and X₁₀, is A, optionally with 1, 2, 3, or 4conservative amino acid substitutions.
 24. The polypeptide of claim 23,wherein the amino acid sequence comprises LKGI (SEQ ID NO:5).
 25. Thepolypeptide of claim 23, wherein the amino acid sequence comprises WLKGI(SEQ ID NO:7).
 26. The polypeptide of claim 23, wherein the amino acidsequence comprises LKGIL (SEQ ID NO:6).
 27. The polypeptide of claim 23,wherein the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95%identity to a sequence of SEQ ID NO:1.
 28. (canceled)
 29. Thepolypeptide of claim 23, wherein the amino acid sequence is SEQ ID NO:2.30. The polypeptide of claim 23, wherein the amino acid sequence has atleast 70%, 80%, 85%, 90%, or 95% identity to a sequence of SEQ ID NO:3.31. (canceled)
 32. (canceled)
 33. The polypeptide of claim 23, whereinthe amino acid sequence is SEQ ID NO:4.
 34. The polypeptide of claim 23,wherein the recombinant polypeptide comprises at least 10 amino acids,15 amino acids, or 20 amino acids and no more than 100 amino acids. 35.A composition for treatment of skin comprising at least one isolated,synthetic, or recombinant polypeptide of claim 23 and a therapeutic,nutraceutical, or cosmetic excipient.
 36. The composition of claim 35,wherein the excipient is configured for topical application. 37.(canceled)
 38. The composition of claim 36, wherein the composition is acream, an ointment, a gel, a liquid, an oil, a powder, a lotion, aserum, an emulsion, a moisturizer, a foam, a face mask, a mousse, anaerosol, a spray, a cleanser, a toner, a topical patch, or a shampoo.39. (canceled)
 40. (canceled)
 41. The composition of claim 35, whereinthe composition comprises at least one skin hydrating agent selectedfrom a group consisting of glycerin, squalene, sorbitol, hyaluronicacid, hyaluronic acid derivatives, sodium hyaluronate, sodiumhyaluronate crosspolymer, niacinamide, glycoproteins, pyrrolidonecarboxylic acid (PCA), lysine HCl, allantoin and algae extract.
 42. Thecomposition of claim 35, wherein the composition comprises at least oneemollient selected from a group consisting of plant oils, mineral oil,shea butter, cocoa butter, petrolatum, fatty acids, triglycerides,benzoates, myristates, palmitates, stearates, glycolipids,phospholipids, squalene, glycerin, ceramide, and algae extract. 43.(canceled)
 44. The composition of claim 35, wherein the compositioncomprises at least one vitamin selected from a group consisting ofvitamin A, vitamin D, vitamin E, vitamin F, vitamin K, vitamin B1(thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5(pantothenic acid), vitamin B7 (biotin), vitamin B6, vitamin B12(cyanocobalamin), vitamin B9, folic acid, niacinamide, or derivativesthereof.
 45. The composition of claim 35, wherein the compositioncomprises an effective amount of the isolated, synthetic, or recombinantpolypeptide of about 500 nM to about 500 μM.
 46. The composition ofclaim 35, wherein the composition comprises an effective amount of theisolated, synthetic, or recombinant polypeptide of about 0.001% to about5% (w/w).
 47. (canceled)
 48. The composition of claim 35, wherein thecomposition improves at least one of skin moisture content,trans-epidermal water loss (TEWL), dermal thickness and echogenicity,intracutaneous analysis, skin viscoelastic properties, or skin surfaceprofile of skin of a user after using composition on the skin ascompared to before using the composition.
 49. The composition of claim35, wherein the composition reduces appearance of lines/wrinkles,appearance of skin tone (evenness), appearance of pores, appearance oftexture/smoothness, firmness (visual), elasticity (tactile), epidermalbarrier, skin roughness, skin hyperpigmentation, or overall appearanceof skin of a user after using composition on the skin as compared tobefore using the composition.
 50. The composition of claim 35, whereinthe treatment of skin with the composition reduces or treats an effectof aging on the skin.
 51. The composition of claim 35, wherein thetreatment of skin with the composition reduces an effect of cellularsenescence on the skin.
 52. The composition of claim 50, wherein theeffect of aging is assessed by at least one of senescence associatedB-galactosidase activity level, a ratio of ATRX foci/cell, p16expression, IL-8 expression, Ki-67 expression, hyaluronic synthase 2expression, matrix metalloprotease 1 (MMP1) expression, sirtuin 6(SIRT6) expression, BLM expression, or exonuclease 1 (EXO1) expression.53.-85. (canceled)